Role of ribosomal proteins eL40 and eS12 in the biogenesis and function of yeast ribosomes

! Departamento de Genética Universidad de Sevilla Role of ribosomal proteins eL40 and eS12 in the biogenesis and function of yeast ribosomes Memoria presentada para aspirar al grado de Doctor en Biología V.o B.o: El director de la Tesis El doctorando Jesús de la Cruz Díaz Sara Martín Villanueva ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! ! This! work! was! funded! by! a! FPI! fellowship! from! the! Spanish! Ministry! of! Economy!and!Competitiveness!(BESA2014A069646),!by!project!grants!from! the!Spanish!Government!(BFU2013A42958AP!and!BFU2016A75352AP),!and!a! shortAterm! fellowship! from! the! Spanish! Ministry! of! Economy! and! Competitiveness!(EEBBAIA2017A12401).! ! A mis abuelos, por creer siempre en mi. ! INDEX ! 1.(RESUMEN!..........................................................................................................................!1( 2.(INTRODUCTION!................................................................................................................!5! • The!ribosome:!Composition,!evolution!and!function!................................................!7! • Ribosomal!proteins!in!S.#cerevisiae!...........................................................................!9! • Ribosome!biogenesis!in!S.#cerevisiae!......................................................................!11! • Transcription!and!processing!of!preArRNAs!........................................................!13! • Assembly!of!the!SSU:!Binding!and!dissociation!of!the!90S!preAribosome!..........!16! • Assembly!of!the!SSU:!Nucleocytoplasmic!stages!................................................!18! • Nucleolar!and!nucleoplasmic!assembly!of!the!large!rAsubunit!...........................!20! • Cytoplasmic!stages!of!the!large!rAsubunit!..........................................................!23! • Folding,!transport!and!degradation!of!ribosomal!proteins!.....................................!24! • Ubiquitin!and!ubiquitinAlike!proteins!......................................................................!27! • Ubiquitination!and!deubiquitination!..................................................................!29! • Functions!of!ubiquitination!and!the!ubiquitin!code!...........................................!31! • Ubiquitin!synthesis:!ubiquitin!genes!...................................................................!33! • Role!of!ubiquitin!in!rAproteins!eL40!and!eS31!....................................................!36! 3.(OBJECTIVES!.....................................................................................................................!38! 4.(RESULTS!..........................................................................................................................!43! CHAPTER(1(–(The(ubiquitin(moiety(of(Ubi1(is(required(for(proper(production( of(ribosomal(protein(eL40(in(Saccharomyces+cerevisiae!...................................................!45! • INTRODUCTION!.......................................................................................................!47! • RESULTS!...................................................................................................................!53! • Generation!of!ubi1!mutants!for!the!phenotypic!analysis!of!the!ubiquitinA L40A!fusion!protein!...........................................................................................!53! • The! ubi1∆ub! ubiquitin! deletion! mutant! displays! a! slowAgrowth! phenotype!.........................................................................................................!54! • The!SMT32S2eL40S!ubi2∆!mutant!displays!a!slowAgrowth!phenotype!..............!56! • Role!of!the!ubiquitin!and!SUMO!moieties!on!the!expression!levels!of! eL40A!.................................................................................................................!57! • The! genomic! ubi1∆ubAHA! and! SMT32S2eL40AAHA! mutations! affect! 60S!rAsubunit!biogenesis!and!translation!..........................................................!58! • Expression!in#trans!of!free!ubiquitin!fails!to!rescue!the!deficiencies! of!the!ubi1∆ubAHA!ubi2∆!and!SMT32S2eL40AAHA!ubi2∆!mutants!.....................!61! • Ubiquitin! and! SUMO! modestly! prevent! eL40! or! S31! protein! aggregation!.......................................................................................................!63! • DISCUSSION!.............................................................................................................!67! • MATERIALS!AND!METHODS!....................................................................................!71! CHAPTER(2(–(Role(of(the(beak(ribosomal(protein(eS12(in(ribosome(biogenesis( and(function(in+Saccharomyces+cerevisiae!........................................................................!77! • INTRODUCTION!.......................................................................................................!79! • RESULTS!...................................................................................................................!83! • Loss!of!eS12!results!in!a!severe!growth!defect!.................................................!83! • Loss!of!eS12!leads!to!a!strong!40S!rAsubunit!shortage!......................................!85! • Deletion!of!RPS12!impairs!rRNA!processing!......................................................!85! • The!rps12∆!deletion!mutant!accumulates!the!20S!preArRNA!in!the! cytoplasm!..........................................................................................................!89! • The!accumulated!20S!preArRNA!is!not!efficiently!incorporated!into! translating!ribosomes!in!rps12∆!cells!................................................................!91! • Dynamics! of! the! transAacting! factors! Enp1! and! Ltv1! during! 40S! rA subunit!biogenesis!in!the!rps12∆!and!ubi3∆!mutants!.......................................!94! • The! absence! of! eS12! or! eS31! induces! an! increase! in! translational! misincorporation!...............................................................................................!98! • DISCUSSION!...........................................................................................................!101! • MATERIALS!AND!METHODS!..................................................................................!105! 5.(GENERAL(DISCUSSION!..................................................................................................!113! ! 6.(CONCLUSIONS!...............................................................................................................!120! 7.(BIBLIOGRAPHY!..............................................................................................................!124! ! 2.(INTRODUCTION( ! ! ! 7( Introduction THE(RIBOSOME:(COMPOSITION,(EVOLUTION(AND(FUNCTION( Ribosomes! are! the! molecular! organelles! that! universally! catalyse! the! protein! synthesis,!process!also!known!as!translation.!During!translation,!the!messenger!RNA! (mRNA)! serves! as! a! template! to! produce! proteins.! Hence,! ribosomes! are! essential! organelles!with!an!important!task!in!gene!expression:!the!decoding!of!the!genetic!code! in!all!organisms!(Wool,!1979).! Mature!ribosomes!consist!of!two!subunits,!one!large!(LSU)!and!one!small!(SSU),! which! both! are! composed! of! ribosomal! RNAs! (rRNAs)! and! proteins! (rAproteins)! associated!through!a!network!of!intermolecular!bridges.!Both!rAsubunits!fulfil!different! tasks!in!protein!synthesis:!the!LSU!catalyses!the!peptide!bond!formation!and!the!SSU!is! responsible! for! the! decoding! process! (Schmeing! and! Ramakrishnan,! 2009).! In! prokaryotes,!the!LSU!and!SSU!are!also!called!50S!and!30S,!respectively;!the!interaction! of!these!subunits!forms!the!70S!ribosomes!(Davis!and!Williamson,!2017).!In!turn,!in! eukaryotes,!mature!ribosomes!known!as!80S!are!composed!of!large!60S!subunits!and! small!40S!subunits!(Figure(1).!Furthermore,!mitochondria!and!chloroplasts!have!their! own!ribosomes.!In!the!budding!yeast!Saccharomyces#cerevisiae,!the!eukaryote!model! organism!used!in!this!Thesis!work,!the!LSU!is!composed!of!rRNAs!25S,!5.8S!and!5S!and! 46!rAproteins,!whereas!the!SSU!consists!of!the!rRNA!18S!and!33!rAproteins!(BenAShem! et!al.,!2011).!! Over!the!last!years,!cryogenic!electron!microscopy!(cryoAEM)!and!crystallography! studies!have!provided!crucial!information!about!the!structure,!function!and!synthesis! of! ribosomes! (BenAShem! et! al.,! 2011;! Klinge! et! al.,! 2012).! From! the! structures,! we! learnt! that! the! rAproteins! are! mostly! distributed! on! the! solventAsurface! of! the! ribosome,!whereas!the!rRNAs!are!mainly!present!in!the!intersubunit!interface,!where! the!functional!activities!of!the!ribosome!reside!(Wilson!and!Doudna!Cate,!2012).!Four! structural!domains!that!are!sequentially!assembled!are!distinguished!in!the!SSU:!the! body,!the!platform,!the!head!and!the!beak!(Figure(1A).!The!beak!region!is!under!study! in!this!Thesis!work!(see(chapter(2). In!the!LSU!there!is!a!central!protuberance,!where! the!peptidyltransferase!centre!(PTC)!is!present!and!the!L1!and!acidic!rAstalks!(Figure( 1B)((BenAShem! et! al.,! 2011).! Although! apparently! monolithic,! the! LSU! is! really! ! ! 8( Introduction partitioned!into!sections!that!assemble!sequentially. ! The!composition!and!the!structure!of!rAsubunits!are!well!conserved!in!evolution,! being! eukaryotic! ribosomes! larger! and! more! complex! than! those! from! prokaryotes.! Prokaryotic!and!eukaryotic!ribosomes!share!a!common!core,!which!is!in!charge!of!the! fundamental!reactions!during!the!translation!process!(Figure( 2).!However,!there!are! kingdomsAspecific! elements! such! as! different! rAproteins! and! specific! extension! FIGURE(1.(Crystal(structure(of(ribosomal(subunits(from(S.+cerevisiae.((A)!The!subunit!interface!(left)! and!the!solventAexposed!surface!(right)!of!the!small!subunit.!(B)!The!subunit!interface!(left)!and!the! solventAexposed!surface!(right)!of!the!large!subunit.!(C)!Association!of!40S!and!60S!subunits!through! intermolecular!bridges!to!form!the!complete!80S!ribosome.!Both!ribosomal!subunits!are!composed! of! rAproteins! and! rRNAs.! The! 60S! rAproteins! are! coloured! in! orange! and! the! 40S! rAproteins! are! coloured!in!blue.!The!rRNAs!are!coloured!in!grey.!CP,!central!protuberance;!GAC,!GTPaseAactivation! center.!Adapted!from!(de!la!Cruz!et!al.,!2015).( ! ! 9( Introduction segments!in!rRNAs!and!rAproteins!(Melnikov!et!al.,!2012;!Melnikov!et!al.,!2018).!Thus,! ribosomes! grow! in! size! upon! transition! from! bacteria! to! eukaryotes.! Translation! initiation! is! quite! different! between! distinct! kingdoms;! thus,! most! of! eukaryoticA specific!elements!of!the!SSU!are!involved!in!this!process. ! RIBOSOMAL(PROTEINS(IN(S.+cerevisiae+ The!rAproteins!have!some!peculiarities;!generally,!most!rAproteins!are!small,!basic! (except!proteins!of!the!acidic!stalk)!and!have!intrinsically!disordered!domains.!Their! basic!nature!is!important!to!neutralize!the!negative!charges!of!the!rRNAs,!given!that! practically! all! rAproteins! directly! interact! with! rRNA! in! mature! ribosomes.! Mostly,! rA proteins! are! distributed! on! the! solventAsurface! of! the! ribosome! (BenAShem! et! al.,! 2011).!However,!most!eukaryotic!rAproteins!also!contain!long!specific!extensions!that! can! penetrate! deeply! into! the! ribosome! (Figure( 3).! Some! of! these! extensions! are! important!to!stabilize!rRNA!junctions!or!to!mediate!contacts!between!ribosomes!and! FIGURE( 2.( Composition( of( bacterial( and( eukaryotic( ribosomes( and( the( common( core.!The! conserved!ribosomal! core!between!bacteria!and!eukaryotes!is!composed!of!RNA!(light!blue)!and! proteins! (light! red).! DomainAspecific! proteins,! extensions! and! protein! insertions! (dark! red),! and! extension!segments!in!ribosomal! RNA!(dark!blue)!are!shown.!Dashed! lines! correspond! to! flexible! elements!from!higher!eukaryotes.!Adapted!from!(Melnikov!et!al.,!2012).( ! ! 10( Introduction their! ligands! (Melnikov! et! al.,! 2018).! Furthermore,! these! extensions! can! serve! as! nuclear!localization!signals!to!deliver!the!rAproteins!to!the!nucle(ol)us,!where!ribosome! biogenesis!takes!place,!or!as!chaperones!binding!site!to!help!their!transport!through! the! nuclear! pore! complex! (NPC),! offering! simultaneous! protection! predominantly! against!protein!aggregation!(e.g.!(FernándezAPevida!et!al.,!2016)!(Pillet!et!al.,!2015)).!In! mammal!cells,!additional!roles!have!been!described!for!these!extensions.!For!example,! extension! of! uL13! (formerly! L16)! controls! translation! of! inflammatory! mRNAs! (Mazumder! et! al.,! 2003)! and! extensions! of! uL5! and! uL18! (formerly! L11! and! L5! respectively)! control! programmed! cell! death! through! inhibition! of! the! oncoprotein! Mdm2,!which!prevents!ubiquitination!of!p53!(Deisenroth!and!Zhang,!2010).! As! mentioned! before,! there! are! 79! rAproteins! in! S.# cerevisiae;! 59! of! these! rA proteins!are!encoded!by!two!paralogous!genes!(Kellis!et!al.,!2004).!Usually,!the!amino! FIGURE(3.(Ribosomal(proteins(of(the(large(ribosomal(subunit.!The!large!rAsubunit!of!Tetrahymena# thermophila# contains! 42! rAproteins.! Six! of! them! are! eukaryoticAspecific! and! 16! are! present! in! all! domains!of!life.!EukaryoticAspecific!extensions!and!proteins!are!depicted!in!red.!Protein!cores!found! in! all! kingdoms! are! depicted! in! light! blue! and! proteins!with!archaeal! homologs! are! in! gold.!The! positions!of!the!NA! and!CAterminal! ends!are! indicated.!The! human! nomenclature! is!used! and! the! yeast!homolog!is!shown!in!parentheses!when!the!yeast!nomenclature!differs.!Adapted!from!(Klinge! et!al.,!2011).!( ! ! 11( Introduction acid!sequences!from!both!paralogues!are!very!similar!but!not!identical!and!the!effects! of! deleting! one! of! the! two! genes! are! different;! thus,! it! has! been! suggested! the! possibility! of! paralogueAspecific! functions! (Chaillou,! 2019).! Assembly! of! a! specific! paralogue!rAprotein!or!its!counterpart!results!in!heterogeneous!ribosome!populations,! which!have!been!speculated!to!be!able!to!perform!different!cellular!physiological!roles.! Changes!in!the!ribosome!composition!can!occur!at!additional!levels:!posttranslational! modifications! of! rAproteins,! alternative! forms! of! rRNAs! and! postAtranscriptional! modifications!of!rRNAs,!as!well!as!binding!to!other!nonAribosomal!proteins!(Genuth! and! Barna,! 2018).! Given! this! heterogeneity,! the! idea! of! specialized! ribosomes! has! recently! arisen! (Chaillou,! 2019).! This! specialization! could! imply! a! new! level! of! regulation!of!gene!expression.! ! RIBOSOME(BIOGENESIS(IN(S.+cerevisiae( Ribosome!synthesis!is!a!very!important!cellular!task.!Yeast!cells!produce!around! 2000! ribosomes! per! minute! in! exponential! growth! conditions! (Warner,! 1999).! This! process!consumes!a!lot!of!cellular!resources!as!it!requires!the!synthesis!of!rRNAs,!rA proteins!and!the!function!of!76!small!nucleolar!RNAs!(snoRNAs)!and!more!than!200! nonAribosomal! transAacting! proteins,! also! known! as! ribosome! assembly! factors! (Woolford! and! Baserga,! 2013).! These! factors! provide! speed,! accuracy,! timing! and! directionality!to!the!ribosome!synthesis!process!and!participate!in!its!regulation.(! Ribosome!biogenesis!starts!in!the!nucleolus,!where!preArRNAs!are!produced!and! nascent!preAribosomal!particles!are!formed!(Figure( 4).!These!preAribosomal!particles! travel!through!the!nucleoplasm!to!the!cytoplasm,!where!the!last!maturation!steps!take! place! (Woolford! and! Baserga,! 2013).! This! route! is! enormously! dynamic,! since! many! factors! and! proteins! must! associate! and! further! dissociate! upon! performing! their! respective!functions.! Moreover,!a!large!number!of!structural! rearrangements!occur.! Ribosome! biogenesis! must! be! rigorously! regulated! as! defects! in! ribosome! assembly! have!deleterious!consequences.!In!fact,!dysregulation!of!ribosome!assembly!have!been! closely!linked!to!human!diseases!(Teng!et!al.,!2013).! ! ! 12( Introduction Ribosome! production! depends! on! nutrient! availability,! adaptation! to! stress! and!the!capability!to!progress!through!the!cell!cycle.!The!TORC1!and!protein!kinase! A! (PKA)! signalling! pathways! mediate! this! regulation! (Figure( 5).! These! pathways! control! the! level! of! both! preArRNAs! and! rAproteins! transcriptionally! and! postA transcriptionally!(de!la!Cruz!et!al.,!2018).!! The!first!step!in!ribosome!biogenesis!is!the!transcription!of!the!ribosomal!DNA! (rDNA).! In! S.# cerevisiae,! there! are! around! 150! tandem! repeats! of! the! rDNA! transcription! unit! located! in! chromosome! XII! (Figure( 6).! Each! transcriptional! unit! FIGURE( 4.( Eukaryotic( ribosome( assembly( route.!Eukaryotic! ribosome! assembly! begins! with! the! transcription!of!the!rDNA!in!the!nucleolus!by!the!RNA!polymerase!I,!which!transcribes!the!35S!preA rRNA! and! the! RNA! polymerase! III,! which! transcribes! the! 5S! rRNA.! On! the! other! hand,! the! RNA! polymerase!II!synthesizes!the!snoRNAs!and!the!mRNAs!that!translate!into!the!rAproteins.!Ribosome! assembly! factors! and! rAproteins! associate! with! the! nascent! preArRNAs! to! form! preAribosomal! particles.!40S!and!60S!rAsubunits!undergo!apparently! independent!maturation!pathways! in!which! many! different! assembly! factors! transiently! associate.! PreAribosomal! subunits! travel! across! the! nucleoplasm! to! the! cytoplasm,! where! the! last! assembly! factors! dissociated! and! the! mature! 80S! ribosome!acquires!functionality.!Adapted!from!(Bassler!and!Hurt,!2018).( ! ! 13( Introduction contains!the!preArDNA!unit!35S! (that! will! be! processed! to!produce!the!mature!18S,! 5.8S!and!25S!rRNAs)!and!5S!rDNA!unit!(Nomura!et!al.,!2004).!Despite!the!high!copy! number! of! these! repeats,! only! about! half! of! the! 150! rDNA! copies! are! actively! transcribed! simultaneously! under! exponential! growth! conditions,! while! the! other! copies! are! maintained! inactivated.! Growth! conditions! can! modulate! this! proportion! since! rDNA! is! highly! transcribed! when! nutrients! are! abundant! but! certain! environmental!stresses!like!a!depletion!of!nutrients!leads!to!lower!transcription!level! of!rDNA.!The! high! number!of! copies!could! serve! as! a!protection!against! mutations,! thus,!maintaining!genome!integrity!(Ide!et!al.,!2010).! Transcription(and(processing(of(pre]rRNAs! In!yeast,!the!RNA!polymerase!III!(RNAP!III)!transcribes!the!5S!rRNA!in!a!precursor! form.! This! precursor! will! be! processed! exonucleolytically! at! its! 3’! end! by! the! exonuclease!Rex1!and,!this!mature!5S!rRNA!will!be!part!of!the!large!subunit!(Ciganda! FIGURE( 5.( Regulation( of( ribosome( biogenesis( in( S.+ cerevisiae.!The! TORC1! and! PKA! pathways! regulate!ribosome!biogenesis!at!different!levels.!They!control!the!transcription!of!ribosomal!proteins! (RP)!and!ribosomal!biogenesis!(RiBi)!regulons!and!rDNA.!PostAtranscriptionally,!they!regulate! preA rRNA!processing,!ribosome!assembly,!translation!and!the!degradation!of!rAsubunits.!Furthermore,!a! feedback!system!specifically!regulates!rAprotein!and!rRNA!and!ribosome!biosynthesis!(RRB)!genes.! Adapted!from!(de!la!Cruz!et!al.,!2018).( ! ! 14( Introduction and!Williams,!2011).!On!the!other!hand,!RNA!polymerase!I!(RNAP!I)!transcribes!the!35S! preArRNA!species.!This!precursor!contains!the!mature!18S,!5.8S!and!25S!preArRNAs!for! the!large!subunit!(Nomura!et!al.,!2004).!Moreover,!35S!preARNA!contains!external!and! internal!transcribed!spacers!(ETS!and!ITS!respectively)!that!are!exoA!or!endonucleolytic! cleaved.!Figure(7!summarizes!the!preArRNA!processing!pathway.! Processing! of! preArRNA! can! occur! postAtranscriptionally! or! coAtranscriptionally! (FernándezAPevida!et!al.,!2015).!In!the!postAtranscriptional!way,!a!complete!35S!preA rRNA!is!synthesized!as!it!is!present!in!the!early!90S!preAribosomal!particle.!However,!in! exponentially! growing! cells,! about! 70%! of! preArRNAs! undergo! coAtranscriptional! cleavage,!before!transcription!of!35S!preArRNA!is!completed!(Koš!and!Tollervey,!2010).! PreArRNA! processing! starts! in! the! nucleolus,! in! the! context! of! preAribosomal! particles!and!it!is!a!multiAstep!process.!Initially,!the!35S!preArRNA!unit!is!cleaved!at!A0! (to! generate! 33S! preArRNA)! and! A1! (to! generate! 32S! preArRNA)! sites! practically! simultaneously!(Hughes!and!Ares,!1991).!Then,!cleavage!at!site!A2!in!ITS1!produces!20S! and!27SA2!preArRNAs!under!exponential!growth!conditions!(Koš!and!Tollervey,!2010).! Separately,! these! precursors! will! continue! 40S! and! 60S! rAsubunits! maturation! FIGURE(6.!Organization(of(the(rDNA(locus(in(S.+cerevisiae.!About!150!tandem!repeats!of!rDNA!are! located!in!the!chromosome!XII.!Each!unit!is!transcribed!by!the!RNA!polymerase!I!(RNA!pol!I)!as!a!35S! precursor!of!rRNAs.!5S!rRNA!is!independently!transcribed!by!the!RNA!polymerase!III!(RNA!pol!III).! 35S! preArRNA!is!processed!to!generated!the!mature! 18S,!5.8S!and!25S! rRNAs.!These!regions! are! flanked!by! two! external! transcribed!spacers! (5’! ETS! and! 3’! ETS)! and! by! two! internal! transcribed! spacers! (ITS1! and! ITS2).! There! are! two! nonAtranscribed! spacers! (NTS)! that! separated! 5S! and! 35S! regions.!Adapted!from!(Woolford!and!Baserga,!2013).( ! ! 21( Introduction 1999).! During! nucleolar! stages,! the! preArRNA! is! folded! into! a! more! compact! conformation! guided! by! scaffolds! formed! by! several! assembly! factors! like! Rrp5,! Mak21,! Noc2! and! Nop4((Granneman! et! al.,! 2011)! (Hierlmeier! et! al.,! 2012).!Other! assembly!factors!(Npa1,!Npa2,!Rsa8!and!Nop8)!and!RNA!helicases!(Dbp2,!Dbp3,!Dbp6,! Dbp7,! Dbp9! Mak5! and! Prp43)! participate! in! very! early! stages! to! stabilize! and/or! to! remodel! RNAs! or! RNPs! and! stay! associated! with! the! preA60S! rAparticle! until! their! removal!in!the!nucleus!or!the!cytoplasm!(RodríguezAGalán!et!al.,!2013)!(Rosado!et!al.,! 2007)!(Joret!et!al.,!2018).!! The!functional!regions!of!60S!rAsubunits!known!as!the!GTPase!activating!centre! (GAC),! peptidyltransferase! centre! (PTC)! and! the! peptide! exit! tunnel! (PET)! start! to! emerge! in! the! nucleolus! by! the! assembly! of! rAproteins! and! cycles! of! association/release!of!assembly!factors!(Kater!et!al.,!2017).!Finally,!functional!PTC!and! PET!regions!are!constructed!upon!the!transition!of!preA60S!subunits!from!the!nucleolus! to!the!nucleoplasm,!where!more!protein!exchange!events!take!place!to!remove!those! factors!that!block!the!formation!of!the!mature!structure!of!rRNA!helices!(Wu!et!al.,! 2016).!! The!formation!of!the!PTC!and!the!PET!is!coupled!with!the!cleavage!of!ITS2!at!the! C2!site,!which!is!catalysed!by!the!endonuclease!Las1!(Gasse!et!al.,!2015).!Previously,! the! remodelling! of! the! rRNA! domains! near! ITS2! is! required.! It! does! so! by! releasing! nearby! assembly! factors! like! Ytm1! and! Erb1,! which! is! mediated! by! the! AAAAATPase! Rea1!(Miles!et!al.,!2005).!Once!ITS2!is!cleaved!by!Las1,!Nop53!is!positioned!where!Erb1! was!previously!located!and!recruits!the!helicase!Mtr4!(Thoms!et!al.,!2015).!Binding!of! Mtr4!makes!the!7S!preArRNA!accessible!for!channelling!into!the!exosome,!which!finally! processes!ITS2!once!it!is!cleaved!(Fromm!et!al.,!2017).! The!central!protuberance!is!also!stabilized!upon!transition!from!the!nucleolus!to! the!nucleoplasm!(Figure( 10).!This!structure!includes!the!5S!rRNA!and!rAproteins!uL5! and!uL18!(known!as!the!5S!RNP).!However,!it!is!not!clear!when!and!how!the!5S!RNP!is! incorporated! into! early! preA60S! rAparticles.! Some! studies! suggest! that! the! importin! Syo1!helps!the!assembly!of!the!5S!RNP!and!this!complex!is!incorporated!into!the!preA 60S!particle!with!assistance!of!the!assembly!factors!Rpf2!and!Rrs1!(Zhang!et!al.,!2007)! ! ! 22( Introduction (Calviño!et!al.,!2015)!(Kressler!et!al.,!2012).!Before!nuclear!export!of!preA60S!particles,! these! two! assembly! factors! are! released! and! the! 5S! RNP! rotates! 180º! to! reach! its! mature!conformation,!serving!as!a!checkpoint!(Leidig!et!al.,!2014).!Others!structural! rearrangements! occur! in! the! central! protuberance! and! some! factors! like! the! AAAA ATPase!Rea1!stabilize!the!rotated!state!(BarrioAGarcía!et!al.,!2015).!ATP!hydrolysis!by! Rea1!induces!the!release!of!the!GTPase!Nog2,!which!permits!the!association!of!the! export!factor!Nmd3!and!recruits!the!exportin!Crm1/Xpo1!(Matsuo!et!al.,!2014).!Nmd3! aids!the!compaction!of!the!preAribosomal!particle!facilitating!its!translocation!and!it! seems!to!prevent!premature!tRNA!incorporation!(Sengupta!et!al.,!2010).!Others!export! factors! are! also! recruited! to! mediate! the! efficient! translocation! of! preA60S! particles! across!the!NPC,!but!only!if!these!checkpoints!are!passed.!For!example,!release!of!the! assembly!factor!Mrt4!enables!binding!of!the!export!factors!Mex67!and!Mtr2,!which!is! coupled!with!the!maturation!of!the!emerging!P!rAstalk!(Sarkar!et!al.,!2016).! FIGURE( 10.( Model( of( nuclear( pre]60S( r]subunit( maturation( events.( Simplified! representation! of! successive!nuclear!proofreading!steps!for!acquisition!of!export!competent!large!preA60S!rAsubunits.! The!first!particle!contains!the!characteristic!“foot”!structure!(ITS2!in!orange)!and!the!5S!RNP!is!in!its! preAmature!conformation.!Next,!the!exonucleolytic!degradation!of!the!ITS2!and!the!rotation!of!the! 5S!RNP!occurs.!The!AAAAATPase!Rea1!and!the!Rix1!subcomplex!sense!the!correct!5S!RNP!position.! The!ATPase!activity!induces!the!activation!and!release!of!the!GTPase!Nog2,!a!prerequisite!for!the! recruitment! of! the! export! adaptor! Nmd3,! which! is! recognized! by! the! exportin! Crm1/Xpo1! in! a! RanGTPAdependent!manner.!The!export!factor!Mex67–Mtr2!is! recruited!close! to!the!nascent!P!rA stalk.!Adapted!from!(Kressler!et!al.,!2017).( ! ! 23( Introduction Cytoplasmic(stages(of(the(large(r]subunit( Once!preA60S!particles!emerge!in!the!cytoplasm,!the!preArRNA!6S!is!processed!to! the!mature!5.8S! rRNA,! last! rAproteins!are!incorporated! and! the!remaining! assembly! factors!are!dissociated!in!a!coordinated!way.!At!this!stage,!GTPases!and!ATPases!drive! the!release!of!these!factors!in!a!hierarchical!pathway!(Figure(11)!(Peña!et!al.,!2017).! The!first!step!in!the!cytoplasmic!maturation!is!the!recruitment!and!activation!of! the!AAAAATPase!Drg1!by!the!assembly!factor!Rlp24,!which!induces!the!replacement!of! Rlp24!by!eL24,!its!homologous!rAprotein!(Kappel!et!al.,!2012).!Then,!the!maturation! pathway!bifurcates!into!events!centered!at!the!PET!or!at!the!joining!surface.!On!one! hand,!Ssa1A4,!Rei1!and!Jjj1!are!required!for!the!dissociation!of!Arx1!and!Alb1!from!the! PET!(Greber!et!al.,!2016).! FIGURE( 11.( Cytoplasmic( maturation( of( pre]60S( ribosomal( particles.! In! last!steps! of! 60S!subunit! maturation,! adaptors! and! assembly! factors! are! sequentially! released! to! enable! assembly! of! the! remaining!rAproteins.! Note!that!the!order!of!some! steps!is! approximate.!Finally,!release!of!Nmd3! allows! the! 60S! rAsubunits! to! gain! translation! competence.! In! the! last! step,! 60S! rAsubunits! gain! translation!competence!by!release!of!Nmd3!and!finally,!acidic!rAproteins!P1!and!P2!assemble!when! both!mature!ribosomal!subunit!are!joined!and!committed!to!translation.!Adapted!from!(de!la!Cruz!et! al.,!2015).( ! ! 24( Introduction On! the! other! hand,! last! steps! on! the! joining! face! are! unclear! since! there! are! controversial!studies.!Some!studies!suggest!that!the!release!of!Nog1!is!coupled!to!the! release!of!Drg1!(Loibl!et!al.,!2014).!However,!recent!results!indicate!that!the!release!of! either! factor! is! uncoupled! from! the! other! and! that! the! NAterminus! of! the! export! adaptor!Nmd3!could!activate!the!release!of!Nog1!(Zhou!et!al.,!2019).!Once!Nog1!is! released,!Nmd3!helps!the!incorporation!of!the!rAprotein!uL16!(formerly!L10),!which!in! turn! is! necessary! for! the! release! of! Nmd3.! Moreover,! timing! of! the! binding! of! the! biogenesis!factor!Yvh1!is!unclear.!This!factor!promotes!P!stalk!assembly!by!displacing! Mrt4;!it!has!been!proposed!that!Yvh1!is!required!in!the!nucleoplasm!for!recruitment!of! the! export! factors! Mex67AMtr2,! previously! to! the! nuclear! preA60S! rAparticle! export! (Sarkar!et!al.,!2016).!However,!recent!findings!suggest!that!binding!of!Yvh1!into!preA 60S!rAparticles!is!blocked!until!Nog1!is!released!in!the!cytoplasm!(Zhou!et!al.,!2019).! Finally,! some! factors! are! recruited! at! the! P! stalk! to! testAdrive! before! starting! the! translation!process.!Incorporation!of!the!rAprotein!uL16!activates!the!GTPase!Efl1!to! release!Tif6!(eIF6),!which!is!a!preArequisite!for!subunit!joining!(Ma!et!al.,!2017).!If!these! cytoplasmic! steps! have! been! completed! successfully,! functional! centres! could! participate!in!protein!synthesis.! ! FOLDING,(TRANSPORT(AND(DEGRADATION(OF(RIBOSOMAL(PROTEINS( Ribosomal! proteins! are! synthetized! in! the! cytoplasm! but! most! of! them! are! incorporated! into! nucle(ol)ar! preAribosomal! particles,! so! they! have! to! travel! to! the! nucleus!to!reach!their!assembly!sites.!However,!as!mentioned!before,!rAproteins!have! particular!features:!most!of!them!are!basic,!contain!intrinsically!disorder!domains!and! long! extensions! rich! in! lysine! and! arginine! (Jäkel! et! al.,! 2002).! Due! to! these! characteristics,!rAproteins!have!a!high!tendency!to!aggregate.!For!this!reason,!different! cellular! mechanisms! have! evolved! to! protect! newly! synthesized! rAproteins! from! aggregation!and!to!ensure!their!safe!delivery!(Peña!et!al.,!2017)!(Pillet!et!al.,!2017).! In! eukaryotes,! molecular! chaperones! are! involved! in! the! folding! of! nascent! proteins! (de# novo! folding).! A! group! of! these! molecular! chaperones! interact! with! ! ! 25( Introduction ribosomes,! binding! close! to! the! PET! and! includes! two! distinct! ribosomeAassociated! chaperone! categories:! the! nascent! polypeptideAassociated! complex! (NAC)! and! the! Hsp70•Hsp40Abased!ribosomeAassociated!complex!(RAC)!(Albanèse!et!al.,!2006).!In!S.# cerevisiae,!RAC!is!composed!by!the!catalyticallyAinactive!Hsp70!homolog!Ssz1!and!the! Hsp40!protein!Zuo1.!RAC!heterodimer!binds!to!the!Hsp70!paralogue!Ssb1!or!Ssb2!to! form!the!chaperone!triad!or!SsbARAC!complex!(Zhang!et!al.,!2017).!In!this!complex,!Ssb! binds!the!nascent!polypeptides!to!their!proper!coAtranslational!folding!and!RAC!acts!as! a!regulator!of!the!process!by!stimulating!the!ATPase!activity!of!Ssb!(Leidig!et!al.,!2013).! Most!rAproteins!can!be!folded!by!this!general!chaperone!system!(Figure(12A).!Previous! studies!have!revealed!that!these!ribosomeAanchored!chaperone!systems!play!a!crucial! role!in!stabilizing!the!aggregationAprone!species!of!rAproteins!in!the!cytoplasm!since! the!simultaneous!deletion!of!NAC!and!SSB/RAC!results!in!the!aggregation!of!more!than! 50!rAproteins!(Koplin!et!al.,!2010).! FIGURE( 12.( Models( for( ribosomal( proteins( folding.( (A)!Scheme! of! the! dual! system! nascent! polypeptideAassociated! complex! (NAC)! and! the! ATPAdependent! Hsp70! SSB/ribosomeAassociated! complex!(SSB/RAC).!Both!machineries!associate!with!ribosomes!and!contact!with!emerging!nascent! polypeptides!(black!line).!These!ribosomeAassociated!chaperones!are!implicated!in!de#novo!folding!of! rAproteins.! Adapted! from! (Koplin! et! al.,! 2010)!(B)!Model! for! the! coAtranslational! capturing! of! ribosomal!proteins!by!their!specific!dedicated!chaperone.!Some!dedicated!chaperones!interact!coA translationally! to! their!specific!rAprotein!partner!proteins!to!prevent!their!aggregation.!Moreover,! dedicated!chaperones!can!promote!nuclear! import!and/or!assist! assembly!of!rAproteins!into!preA ribosomal!particles.!Adapted!from!(Pausch!et!al.,!2015).!( ! ! 26( Introduction Apart! from! these! general! chaperone! machineries,! importins! also! stabilize! rA proteins.!Importins!are!a!group!of!proteins!that!mediate!the!nuclear!import!of!proteins! by!recognition!of!their!nuclear!localization!signals!(NLS).!In!yeast,!principal!importins! that! transport! rAproteins! are! karyopherins! Kap104,! Kap123! and! Pse1! (Rout! et! al.,! 1997).! For! rAproteins,! due! to! their! high! tendency! to! aggregate,! importins! are! also! required!to!act!as!chaperones!protecting!them!from!aggregation!(Jäkel!et!al.,!2002).! However,!importins!release!their!cargo!into!the!nucleus!so!further!mechanisms!may!be! required!to!ensure!protection!of!rAproteins!until!they!reach!their!assembly!site.!! Apparently,!these!mechanisms!are!not!enough!to!ensure!optimal!expression!of!rA proteins,! and! indeed,! specific! rAproteins! require! exclusive! systems.! Recently,! a! new! group! of! proteins! called! dedicated! chaperones! has! been! uncovered.! These! proteins! interact! specifically! with! newly! synthesized! rAproteins! to! prevent! their! aggregation,! promote!their!nuclear!import!and/or!assist!their!assembly!into!preAribosomal!particles! (Pillet!et!al.,!2017).!Until!today,!dedicated!chaperones!for!eleven!rAproteins!have!been! found:!uS3,!uS5,!eS6,!uS11,!eS26,!uL3,!uL4,!uL14,!uL16,!uL5!and!uL18,!but!more!are! expected!to!be!discovered.!In!some!cases,!dedicated!chaperones!can!bind!to!their!rA protein! partners! coAtranslationally! until! the! rAprotein! assembles! into! preAribosomal! particles!(Figure(12B)!(Pausch!et!al.,!2015).!However,!a!class!of!dedicated!chaperones! termed! “escortins”! only! bind! to! their! rAprotein! partners! after! their! import! into! the! nucleus!and!guide!them!through!the!nucleus!to!their!assembly!site!(Pillet!et!al.,!2017).! For!example,!the!escortin!Tsr2!associates!with!eS26!after!its!nuclear!import,!thereby! dissociating!the!eS26Aimportin!complex!in!a!RanGTPAindependent!manner,!and!assists! in!its!assembly!into!90S!preAribosomal!particles!(Schütz!et!al.,!2014).! Interestingly,! unassembled! rAproteins! are! rapidly! degraded! to! maintain! their! cellular! homeostasis.! Recently,! a! degradation! pathway! termed! ERISQ,! for! Excess# RIboSomal#protein#Quality#control,!has!been!described!in!which!the!HECTAdomain!E3! ubiquitin! ligase! Tom1! participates! (Sung! et! al.,! 2016a).! It! seems! that! Tom1! ubiquitinates!residues!of!unassembled!rAproteins!that!are!normally!inaccessible!as!they! are! concealed! in! the! mature! ribosome.! This! type! of! ubiquitination! targets! these! rA proteins! for! proteasomeAdependent! degradation.! Other! mechanisms! are! related! to! ! ! 27( Introduction ribosome!metabolism;!thus,!in!stress!conditions,!40S!rAsubunits!are!located!in!stress! granules! (known! as! PAbodies)! and! they! are! degraded! by! autophagy! (Grousl! et! al.,! 2009).!Moreover,!upon!nutrient!starvation,!mature!ribosomes!are!selectively!degraded! by! a! process! regulated! by! ubiquitination! and! referred! to! as! ribophagy! (Kraft! et! al.,! 2008).!! ( UBIQUITIN(AND(UBIQUITIN]LIKE(PROTEINS( Proteins!are!subject!to!a!high!variety!of!postAtranslational!modifications.!Among! these!protein!modifiers,!one!of!the!most!studied!is!ubiquitin.!Ubiquitin!is!a!small!and! globular!protein!of!76!amino!acids!present!in!all!eukaryotes.!Actually,!ubiquitin!was! originally! named! “ubiquitous! immunopoietic! polypeptide”! and! is! one! of! the! most! highly! conserved! eukaryotic! proteins! known! (Goldstein! et! al.,! 1975).!The! most! common! function! of! ubiquitin! is! tagging! proteins! for! proteasomeAmediated! degradation.! The! proteasome! is! a! large! multiAsubunit! complex! of! proteases! and! regulatory! proteins! shaped! as! a! capped! cylinder! and! inside! of! which! proteins! are! digested!(Finley! et! al.,! 2012).! However,! ubiquitin! can! also! act! as! a! nonAproteolytic! signal! in! the! regulation! of! diverse! cellular! processes! including! DNA! repair,! transcription,!and!trafficking!of!membrane!proteins!(Kwon!and!Ciechanover,!2017).!! Several! other! proteins! structural! and! evolutionarily! related! to! ubiquitin! have! been!discovered.!Like!ubiquitin,!these!proteins!contain!a!βAgrasp!fold!and!are!known! as!ubiquitinAlike!proteins!(UBLs)!(Cappadocia!and!Lima,!2018).!Some!of!these!proteins! are! able! to! be! conjugated! to! others! proteins,! in! a! similar! way! as! ubiquitin.! The! functions! of! UBLs! include! diverse! processes! such! as! nuclear! protein! transport,! DNA! repair,!autophagy,!chromosome!replication,!etc.! Despite!ubiquitin,!UBLs!are!present!not!only!in!eukaryotes!but!have!also!been! found!in!prokaryotes!(Iyer!et!al.,!2006).!However,!not!all!UBL!families!are!found!in!all! eukaryotes.!In!yeast,!several!UBLs!have!been!described!(Figure(13),!for!example,!Smt3,! Rub1,! Urm1,! which! drive! modifications! known! as! SUMOylation,! NEDDylation! and! urmylation,!respectively,!or!Atg8!and!Atg12,!factors!that!participate!in!autophagy.!The! ! ! 28( Introduction UBLAtargets! are! not! always! proteins;! for! example,! Atg8! is! conjugated! to! the! phospholipid!phosphatidylethanolamine!(Ichimura!et!al.,!2000),!and!Urm1!can!act!as!a! sulphur!carrier!in!a!tRNA!modification!pathway!(Wang!et!al.,!2011).!! Among!these!UBLs,!the!SUMO!(Small!UbiquitinAlike!MOdifier)!family!has!gained! importance!in!last!years.!SUMO!is!highly!conserved!in!all!eukaryotes!but!the!number!of! SUMO!genes!is!variable.!For!example,!in!S.#cerevisiae,!only!one!essential!SUMO!gene,! SMT3,! has! been! identified,! while! in! humans! at! least! four! SUMO! genes! have! been! found.! Structurally,! the! main! difference! between! an! ubiquitin! and! a! SUMO! molecule! resides!in!a!NAterminal!extension!of!the!SUMO!protein!that!is!not!present!in!ubiquitin.! The!sumoylation!process!is!analogous!to!the!ubiquitination!one!but!the!enzymes!that! participate! in! both! processes! are! different.! Like! ubiquitination,! sumoylation! is! a! reversible! process.! SUMO! deconjugation! (desumoylation)! is! catalysed! by! ULP! proteases,!which!in!yeast!are!known!as!Ulp1!and!Ulp2!(Cappadocia!and!Lima,!2018).! SUMO!is!involved!in!various!cellular!processes,!such!as!nuclear!transport,!cellA cycle! progression,! protein! trafficking,! stress! response,! etc.! Rather! than! promoting! protein! degradation! like! the! ubiquitin! moiety,! the! SUMO! attachment! blocks! the! proteasomal! degradation! by! competing! with! ubiquitination! (Wilson,! 2017).! Thus,! FIGURE( 13.( Ubiquitin]like( domains.( !Structural! models! of! the! ubiquitinAlike! domain! of! different! proteins!from!S.#cerevisiae.#The!conserved!βAgrasp!fold!is!depicting!in!all!cases.!Adapted!from!(Yu!et! al.,!2016).( ! ! 29( Introduction sumoylation!seems!to!enhance!protein!stability.!However,!another!class!of!enzymes! called!SUMOAtargeted!ubiquitin!ligase!(STUbL)!have!been!discovered.!These!enzymes! recognize!sumoylated!proteins!as!targets!for!ubiquitination.!This!activity!results!in!a! hybrid!signal,!indicating!that!SUMO!and!ubiquitin!can!cooperate!with!each!other!(Nie! and!Boddy,!2016).!! There! are! also! proteins! that! contain! UBL! domains! as! part! of! its! primary! sequence.!These!UBL!domains!are!not!usually!processed!and!confer!properties!similar! to!those!from!a!transferable!UBL,!being!especially!important!to!establish!or!maintain! specific! protein! interactions! (Hochstrasser,! 2000).! Interestingly,! some! ribosome! assembly! factors! harbour! UBL! domains! important! for! ribosome! biogenesis.! For! example,!the!preA60S!assembly!factors!Rsa4!and!Ytm1!contain!NAterminal!UBL!domains! that!directly!interact!with!the!factor!Rea1.!These!associations!are!required!for!the!ATPA dependent!dissociation!of!some!preAribosomal!factors!(Ulbrich!et!al.,!2009).! Ubiquitination(and(deubiquitination( As!stated!above,!ubiquitin! can! be! reversibly! conjugated!to! proteins!as! a! postA translational!modifier!in!a!process!called!ubiquitination.!The!ubiquitination!reaction!is! mediated!by!the!cooperative!action!of!three!types!of!enzymes:!the!ATPAdependent!E1! ubiquitinAactivating!enzyme,!the!E2!ubiquitinAconjugating!enzyme,!and!the!E3!ubiquitin! ligase!(Figure(14)((Finley!et!al.,!2012;!Pickart,!2001).!Commonly,!ubiquitination!results! in!the!formation!of! an!isopeptide! bond! between!the!CAterminal!glycine!(Gly)!of!the! ubiquitin!and!the!amino!group!of!a!specific!lysine!(Lys)!of!the!target!protein!(Pickart,! 2001).! Ubiquitin!itself!has!seven!lysine!residues!(K6,!K11,!K27,!K29,!K33,!K48,!and!K63),! all! of! which! can! be! conjugated! to! a! second! ubiquitin! molecule! (Peng! et! al.,! 2003),! leading!to!the!formation!of!topologically!distinct!polyubiquitin!chains.!Alternatively,!in! the!NAterminal!methionine!(M1),!an!ubiquitin!molecule!can!also!be!attached,!at!least! in!higher!eukaryotes!(Fennell!et!al.,!2018).!! In! yeast,! there! is! only! one! E1! enzyme! (Uba1)! responsible! for! activation! of! ubiquitin.! However,! there! are! multiple! E2! and! E3! enzymes! that! generate! ubiquitin! ! ! 30( Introduction chains.!These!enzymes!determine!the!architecture!of!the!ubiquitin!chain!and!define! the!linkage!type!(Finley!et!al.,!2012).!Regarding!the!diversity!of!possible!combinations,! many!receptors!with!several!ubiquitinAbinding!domains!(UBDs)!have!been!described.! These!receptors!play!a!key!role!in!decoding!ubiquitin!signals!(Kwon!and!Ciechanover,! 2017).! Like!other!postAtranslational!modifications,!ubiquitination!is!a!reversible!process.! Removal!of!ubiquitin!can!be!performed!in!a!single!step!by!deubiquitinating!enzymes! (DUBs).!These!enzymes!hydrolyse!the!linkage!between!ubiquitin!and!their!substrates,! as!well!as!between!ubiquitin!molecules!themselves!(Finley!et!al.,!2012).!In!yeast,!20! DUBs!have!been!identified!but!this!number!varies!in!different!organisms,!for!example,! there! are! around! 100! DUBs! in! humans.! Despite! the! certain! degree! of! redundancy! found!among!DUBs,!there!are!mechanisms!of!regulation! by!which!some!DUBs!have! highly!diversified!functionality!and!require!substrate!specificity,!for!example!depending! on!the!recognition!of!the!ubiquitin!chain!(Mevissen!and!Komander,!2017).!! Deubiquitination!is!necessary!to!recycle!ubiquitin!since!DUBs!recover!ubiquitin! molecules! from! the! modified! proteins.! Therefore,! action! of! DUBs! is! required! to! maintain! the! levels! of! free! ubiquitin! in! the! cell.! Indeed,! defects! in! deubiquitination! cause! reduced! ubiquitin! levels! (Finley! et! al.,! 2012).! Hence,! deubiquitinases! are! key! FIGURE(14.(Scheme(of(the(ubiquitination(process.!Ubiquitin!conjugation!involves!the!cascade!of!the! E1–E2–E3! enzymes.! This! process! can! generate! monoubiquitinated! proteins! or! multiA! or! polyA ubiquitinated! proteins! if! more! than! one! round! of! ubiquitination! are! performed.! Adapted! from! (Finley!et!al.,!2012).( ! ! 37( Introduction Previous!studies!suggest!that!the!proteolytic!maturation!of!these!Ubi!precursors! occurs!very!rapidly,!likely!coAtranslationally;!indeed,!the!precursor!proteins!could!never! be!detected!under!wildAtype!conditions!(FernándezAPevida!et!al.,!2016;!Finley!et!al.,! 1989).!We!have!previously!demonstrated!that!the!proteolytic!maturation!of!the!Ubi! precursors!is!required!for!proper!assembly!of!rAproteins!eS31!and!eL40!into!40S!and! 60S! rAsubunits,! respectively! since! nonAcleaved! Ubi3! or! Ubi1! variants! confer! lethal! phenotypes!when!expressed!as!the!sole!source!of!eS31!or!eL40!(Lacombe!et!al.,!2009)! (MartínAVillanueva!et!al.,!2019).!Moreover,!nonAcleaved!variants!of!Ubi3!or!Ubi1!can! be!incorporated!into!preAribosomal!particles!only!when!there!is!no!competition!with! the!processed!wildAtype!rAproteins.!These!studies!also!indicate!that!Ubi3Acontaining!rA particles!are!slightly!impaired!in!translation!initiation,!whereas!the!Ubi1Acontaining!rA particles! delay! their! cytoplasmic! maturation! and! compromise! the! efficiency! of! translation!elongation;!this!is!probably!due!to!the!fact!that!the!unprocessed!ubiquitin! could!be!interfering!with!the!recruitment!and!function!of!the!elongation!factors!eEF1A! and! eEF2,! which! bind! in! close! proximity! to! eL40! (Lacombe! et! al.,! 2009)! (MartínA Villanueva!et!al.,!2019).! We! have! also! studied! the! consequences! of! deleting! the! NAterminal! ubiquitin! moiety! of! the! Ubi3! precursor.! The! ubiquitin! deletion! mutant! ubi3∆ub!shows! slowA growth!phenotype,!a!shortage!of!40S!rAsubunit!and!18S!rRNA!content!defects,!which! are! suppressed! by! its! increased! dosage! (Finley! et! al.,! 1989;! Lacombe! et! al.,! 2009).! These!results!reveal!that!the!ubiquitin!moiety!is!important!but!not!essential!for!the! functional! integrity! of! eS31.! Accordingly,! it! has! been! proposed! that! the! ubiquitin! moiety!could!act!as!a!chaperone!to!facilitate!the!correct!folding,!thus,!contributing!to! the!efficient!synthesis!of!rAproteins!eS31!and!eL40.!In!agreement!with!this!hypothesis,! practically!all!rAproteins!tend!to!aggregate!in!the!absence!of!the!ribosomeAassociated! SsbARAC!and!NAC!chaperone!complexes,!with!the!apparent!exception!of!eS31!and!eL40! (Koplin!et!al.,!2010).!Additionally,!the!proteolytic!removal!of!ubiquitin!from!eS31!and! eL40!could!serve!to!couple!the!supply!of!ubiquitin!to!the!production!and/or!function!of! ribosomes,!integrating!and!coordinating!the!synthesis!and!degradation!of!proteins!in! eukaryotes.! One! of! the! objectives! of! this! work! is! to! further! examine! the! effects! of! deleting!the!NAterminal!ubiquitin!moiety!of!the!Ubi1!precursor.! ! ! 3.(OBJECTIVES( ! ! ! ! ! ! ! 41( Objectives ( ! The!aim!of!our!group!is!to!deepen!the!knowledge!on!the!function!of!selected! transAacting! factors! and! rAproteins! from! both! rAsubunits! in! the! ribosome! biogenesis! process!of!the!model!eukaryote!S.#cerevisiae.!Specifically,!in!this!Thesis!Work,!I!have! explored! the! role! of! a! pair! of! particular! rAproteins! eL40! and! eS31,! which! are! synthesized!as!precursors!fused!to!an!ubiquitin!molecule,!in!ribosome!biogenesis!and! function.!The!precise!objectives!of!this!work!are: ! ! 1. Study! the! role! of! the! ubiquitin! moiety! fused! to! the! eL40! rAprotein! in! the! biogenesis!of!yeast!ribosomes.!More!specifically,!we!aim!to!analyse!how!the! absence!of!this!cisAacting!ubiquitin!moiety!or!its!replacement!by!the!ubiquitinA like!protein!Smt3!affects!the!expression!and!assembly!of!eL40!in!60S!rAsubunits.! ! 2. Assess!the!contribution!of!the!soAcalled!beak!region!of!40S!rAsubunits!in!yeast! ribosome!synthesis!and!function,!through!the!phenotypical!analysis!of!mutants! lacking!the!rAproteins!eS12!and!eS31,!alone!or!in!combination.! ! ! ! 4.(RESULTS( ! ! CHAPTER(1( ( The(ubiquitin(moiety(of(Ubi1(is(required(for(proper(production( of(ribosomal(protein(eL40(in(Saccharomyces+cerevisiae ! ! ! ! ! 53( Chapter 1 RESULTS( ( Generation(of(ubi1(mutants(for(the(phenotypic(analysis(of(the(ubiquitin]L40A(fusion( protein( Yeast!eL40!is! an! essential! rAprotein!of!52! amino! acids!that!is! encoded!by!two! independent!paralogous!genes,!UBI1!(RPL40A;!YIL148W)!and!UBI2!(RPL40B;!YKR094C).! As! previously! mentioned,! in! practically! all! eukaryotes,! eL40! as! well! as! the! rAprotein! eS31! (UBI3! gene),! are! produced! as! CAterminal! parts! of! ubiquitinAfused! precursor! proteins.!We!have!previously!studied!the! effects! of! impairing! the! ubiquitin!removal! within!Ubi1!and!Ubi3!precursors!by!using!mutants!where!the!efficiency!of!this!removal! was!reduced!(Lacombe!et!al.,!2009;!MartínAVillanueva!et!al.,!2019).!However,!to!date,! only! experimental! data! concerning! the! phenotypic! consequences! of! expressing! a! genomic!ubi3∆ub! allele,! which!lacks!the! region!coding! for! the! ubiquitin!moiety,! are! available! (Finley! et! al.,! 1989;! Lacombe! et! al.,! 2009).! To! explore! the! role! of! the! NA terminal! fused! ubiquitin! moiety! within! Ubi1/2! precursors,! we! constructed! two! different!mutants!(Figure(1).!First,!we!generated!a!mutant!replacing!the!endogenous! wildAtype! UBI1! copy! with! an! ubi1∆ub! mutant,! which! harbours! a! deletion! of! the! ubiquitin! moiety! from! the! Ubi1! precursor.! To! allow! efficient! translation! of! the! construct,!an!ATG!start!codon!was!inserted!just!downstream!the!5'!UBI1!untranslated! sequence;! thus,! the! corresponding! eL40A! protein! started! with! an! NAterminal! methionine!not!present!in!the!natural!eL40A.!Second,!we!replaced!the!endogenous! UBI1! allele! with! a! SMT32S2eL40A! allele,! where! the! ubiquitin! coding! region! was! exchanged!with!a!Smt3!(hereafter!SUMO)!coding!region.!To!facilitate!cleavage!of!the! SUMO!moiety,!we!inserted!a!serine!codon!at!the!junction!between!the!coding!regions! of!Smt3!and!eL40A.!In!both!constructs,!as!well!as!in!a!genomic!wildAtype!UBI1!copy,!we! also!added!an!inAframe!CAterminal!single!HA!epitope!for!western!detection.! ! ! ! ! ! ! ! 54( Chapter 1 The(ubi1∆ub(ubiquitin(deletion(mutant(displays(a(slow]growth(phenotype( For! the! functional! analysis! of! the! generated! mutants,! we! first! examined! the! growth!phenotype!associated!with!the!deletion!of!the!ubiquitin!moiety!from!the!Ubi1! precursor!(Figure( 2).!As!eL40!is!encoded!by!two!independent!paralogous!genes,!we! performed!this!analysis!both!in!the!presence!or!the!absence!of!a!functional!eL40B!rA protein! (wildAtype! UBI2!or! ubi2∆! background,! respectively).! As! previously! described! (FernándezAPevida!et!al.,!2012;!MartínAVillanueva!et!al.,!2019),!the!presence!of!the!HA! epitope!slightly!affected!growth!when!eL40AAHA!is!the!sole!source!of!eL40,!especially! at!low!temperatures.!In!agreement,!doubling!times!of!ca.!1.8!h,!2.0!h!and!3.0!h!were! obtained! in! liquid! YPD! at! 30! ºC! for! the! wildAtype! W303A1A! strain,! and! the! isogenic! ubi2∆!and!UBI12HA#ubi2∆!mutants,!respectively.!As!also!discussed!(MartínAVillanueva! et! al.,! 2019),! this! is! likely! due! an! adverse! effect! of! the! extra! amino! acids! on! the! interaction!environment!of!the!CAterminus!of!eL40A!within!the!ribosome.!Figure(2!also! shows!that!deletion!of!the!ubiquitin!moiety!has!no!effect!on!cell!growth!when!the!UBI2! gene! is! present.! However,! the! ubi1∆ub2HA#ubi2∆! mutant,! in! which! the! sole! cellular! source! of! eL40! comes! from! the! singleAcopy! expression! of! a! ubiquitinAfree! eL40A! rA protein,!showed!a!severe!sg!phenotype,!at!all!tested!temperatures.!Compared!with!the! previous!strains,!the!ubi1∆ub2HA#ubi2∆!mutant!doubled!every!5.5!h!in!liquid!YPD!at!30! ºC.! FIGURE(1.(Schematic(representation(of(the(genomic(constructs(used(in(this(study.!Ubi1!consists!of! an!NAterminal!ubiquitin!moiety!fused!to!the!rAprotein!eL40A.!A!peptide!bond!between!the!CAterminal! glycine!of!ubiquitin!(G76)!and!the!NAterminal!isoleucine!of!eL40A!(I77)!connects!the!two!proteins.! Relevant! residues! and! ubi1# mutant! variants! thereof! used! in! this! study! are! also! indicated.! The! relevant!constructs,!precursor!and!mature!protein!names!are!also!indicated.! The!asterisk!denotes! that!the!exact!nature!of!the!mature!proteins!derived!from!these!precursor!proteins!has!not!been! studied;!thus,!the!indicated!mature!proteins!correspond!to!the!most!likely!products.!A!CAterminal! 1xHA!tag!was!added!for!western!detection!of!the!Ubi1!and/or!eL40!protein!variants.( ! ! 55( Chapter 1 It! has! been! previously! shown! that! increased! dosage! of! the! ubi3∆ub! allele! suppresses! its! growth! defect! phenotype! (Finley! et! al.,! 1989;! Lacombe! et! al.,! 2009).! Thus,!to!address!whether!a!similar!situation!occurred!upon!an!increased!dosage!of!the! ubi1∆ub! allele,! we! cloned! either! the! wildAtype! UBI1AHA! or! the! mutant! ub1∆ubAHA! allele!on!YCplac111,!a!lowAcopyAnumber!CEN!LEU2!plasmid!(Gietz!and!Sugino,!1988),! and! transformed! a! shuffle! ubi1∆# ubi2∆! strain! (TAY001! strain;! see! Table( 1).! After! plasmid!pHT4467∆AUBI1!(URA3#ADE3)!shuffling!on!5AFOAAcontaining!plates,!cells!were! analysed!on!YPD!plates.!As!shown!in!Figure(2,!expression!of!the!ub1∆ubAHA!allele!from! a!CEN!plasmid!was!able!to!significantly!ameliorate!the!growth!defect!of!the!ubi1∆ub2 HA#ubi2∆!strain!to!apparently!the!wildAtype!extend.!This!is!in!agreement!with!previous! data! showing! that! the! expression! of! a! ubi1∆ub! allele! from! a! strong! promoter! also! complemented!the!lethal!phenotype!of!a!ubi1∆#ubi2∆!strain!to!the!wildAtype!extend! (Finley! et! al.,! 1989).! We! conclude! that! the! ubiquitin! moiety! of! Ubi1! is! required! for! optimal!cell!growth!when!its!expression!is!driven!from!a!singleAcopy!allele.! ! ! FIGURE(2.(The(genomically(integrated(ubi1∆ub6HA+allele(confers(a(slow]growth(phenotype.! Growth! analysis!of! the!indicated!strains,!either!expressing!a!CAterminally!HAAtagged!Ubi1!(UBI12HA)!or!an! Ubi1∆ub!(ubi1∆ub2HA)!variant!protein!from!the!UBI1!genomic!locus!or!from!a!centromeric!plasmid! (+!ubi1∆ub2HA),!in!a!wild!type!UBI2!or!a!ubi2∆!null!mutant!background .(Strains!W303A1A!(Wild!type),! SMY113!(UBI12HA),!SMY256!(UBI12HA!ubi2∆),!SMY107!(ubi1∆ub2HA),!SMY257!(ubi1∆ub2HA!ubi2∆),! SMY215![YCplac111Aubi1∆ubAHA]!(ubi1∆!ubi2∆#+#ubi1∆ub2HA)!and!JDY922!(ubi2∆)! were!spotted!in! fivefold!serial!dilution!steps!onto!YPD!plates,!which!were!incubated!for!4!days!at!22!ºC!or!for!3!days! at!30!ºC!and!37!ºC.!( ! ! 56( Chapter 1 The(SMT36S6eL40S(ubi2∆(mutant(displays(a(slow]growth(phenotype( The! small! ubiquitinAlike! modifier! SUMO! shares! an! overall! threeAdimensional! structure! very! similar! to! ubiquitin! and! possess,! as! ubiquitin,! a! CAterminal! glycine! residue!(e.g.!(van!der!Veen!and!Ploegh,!2012)).!Thus,!we!reasoned!that!if!ubiquitin!had! a! cisAacting! function! affecting! that! of! the! eL40! protein,! the! replacement! of! the! ubiquitin! moiety! in! Ubi1! by! the! SUMO! molecule! should! not! drastically! impair! the! function!of!eL40!if!properly!processed.!To!this!aim,!we!first!analysed!the!consequences! in!growth!of!replacing!the!ubiquitin!moiety!of!the!Ubi1!precursor!by!the!ubiquitinAlike! modifier!SUMO.!As!above,!we!used!both!the!UBI2!and!ubi2∆!backgrounds.!As!shown!in! Figure(3,!when!the!wildAtype!UBI2!allele!is!present,!the!strain!displays!apparent!wildA type!growth.!Interestingly,!the!SMT32S2eL40A2HA!ubi2∆!strain,!where!the!sole!source! of! eL40! comes! from! the! precursor! Smt3ASAeL40AAHA,! shows! a! mild! but! significant! improvement!of!the!cell!growth!if!compared!with!that!of!the!isogenic!ubi1∆ub#ubi2∆! counterpart,! which! is! especially! evident! at! higher! temperatures.! Consistently,! compared!to!other!ubi1!strains,!the!SMT32S2eL40A2HA!ubi2∆!strain!doubled!every!4.0! h!in!liquid!YPD!at!30!ºC.!As!above,!increasing!the!gene!dosage!of!SMT32S2eL40A2HA#by! FIGURE(3.(The(genomically(integrated(SMT36eL40A(allele(permits(better(growth(than(the(ubi1∆ub6 HA+one.( Growth!analysis!of!the!indicated!strains,!either!expressing!a!CAterminally!HAAtagged!Ubi1! (UBI12HA)!or!a!fusion!Smt3AeL40A!(SMT32eL40A2HA)!variant!protein!from!the!UBI1!genomic!locus!or! from! a! centromeric! plasmid! (+! SMT32eL40A2HA),! in! a! wild! type! UBI2!or! a! ubi2∆!null! mutant! background.( Strains! W303A1A! (Wild! type),! SMY218! (SMT3AeL40AAHA),! SMY258! (SMT3AeL40AAHA! ubi2∆),! SMY256! (UBI12HA! ubi2∆),! SMY257! (ubi1∆ub2HA! ubi2∆),! SMY216! [YCplac111ASMT3AeL40AA HA]!(ubi1∆!ubi2∆#+#SMT3AeL40AAHA)!and!JDY922!(ubi2∆)!were!spotted!in!fivefold!serial!dilution!steps! onto!YPD!plates,!which!were!incubated!for!4!days!at!22!ºC!and!for!3!days!at!30!ºC!and!37!ºC.!( ! ! 57( Chapter 1 the!use!of!a!lowAcopyAnumber!plasmid!is!suppressing!the!sg!phenotype!linked!to!the! genomic!singleAcopy!SMT32S2eL40A#allele.#We!conclude!that!SUMO!is!able!to!partially! compensate!the!loss!of!the!cisAacting!role!of!the!ubiquitin!moiety!of!Ubi1!on!eL40A.! ! Role(of(the(ubiquitin(and(SUMO(moieties(on(the(expression(levels(of(eL40A( The!fact!that!the!sg!phenotype!linked!to!the!expression!of!either!the!ubi1∆ub2HA! or! SMT32S2eL40A2HA#alleles! as! the! sole! source! of! cellular! eL40! rAprotein! was! suppressed!by!increasing!the!copyAnumber!of!the!respective!alleles,!strongly!suggests! that!the!ubiquitinAcoding!tail!of!the!UBI1!gene!facilitates!the!expression!of!eL40A!rA protein.!To!test!this!hypothesis,!we!monitored!the!expression!levels!of!the!eL40AAHA! protein!in! the! different! used!strains!by! western! blotting.! As! shown!in!Figure( 4,!the! protein! levels! of! eL40AAHA! significantly! decreased! in! the! ubi1∆ub2HA# ubi2∆! strain,! compared! with! the! expression! of! wildAtype! eL40AAHA! rAprotein! from! the! UBI1AHA! allele.!As!expected,!an!increased!dosage!of!the!ubi1∆ub2HA#allele!restores!the!protein! levels! of! eL40AAHA! (Figure( 4,( lane( 5).! Together,! these! results! indicate! that! the! ubiquitin!moiety!of!Ubi1!is!required!for!the!efficient!production!of!the!eL40!rAprotein.! ! To!assess!the!efficiency!of!Smt3ASAeL40AAHA!cleavage!into!SUMO!and!eL40AAHA,! western! blot! analyses! using! antiAHA! antibodies! were! also! carried! out! in! whole! cell! extracts! of! strains! harbouring! a! genomic! SMT32S2eL40A# allele.! As! previously! shown! (FernándezAPevida!et!al.,!2012;!MartínAVillanueva!et!al.,!2019),!no!Ubi1AHA!precursor! but!only!mature!eL40AAHA!was!detected!in!the!UBI1AHA!strains!(Figure(4,(lanes(2(and( 3).! In! contrast,! although! mature! eL40AAHA! was! fairly! well! detected! in! the! genomic! SMT32S2eL40A2HA#ubi2∆#strain,!some!precursor!protein!could!also!be!detected!at!the! expected!molecular!mass,!especially!when!the!fusion!Smt3ASAL40A!protein!variant!is! the! sole! cellular! source! of! eL40! (Figure( 4,( lanes( 6( and( 7).! We! conclude! that! replacement!of!the!ubiquitin!by!a!SUMO!moiety!followed!by!a!linked!serine!at!its!CA terminus! reduces! the! efficiency! of! the! proteolytic! processing! of! the! precursor! into! SUMO! and! eL40A.! Nevertheless,! a! clear! increase! in! the! total! amount! of! produced! mature!eL40AAHA!is!observed!in!the!cell!extracts!of!the!SMT32S2eL40S#strains.! ! ! 58( Chapter 1 ! The( genomic( ubi1∆ub]HA( and( SMT36S6eL40A]HA( mutations( affect( 60S( r]subunit( biogenesis(and(translation( As! the! genomic! ubi1∆ub2HA! allele! limits! the! production! of! eL40A! protein! in! a! ubi2∆!background,! we!wonder!whether!this! is!also!accompanied!by!a! 60S!rAsubunit! shortage.!Thus,!we!performed!polysome!profile!analyses!in!the!different!ubi1!mutants! described!in!this!study.!We!first!examined!the!profiles!of!the!control!UBI1AHA!ubi2∆! strain!grown!in!YPD!at!30!ºC.!As!shown!in!Figure(5A,!this!strain!showed,!as!expected! for!the!presence!of!the!ubi2∆!allele!(FernándezAPevida!et!al.,!2012),!a!very!mild!deficit! in!60S!rAsubunits!as!evidence!for!the!appearance!of!halfAmer!polysomes.!In!contrast,! the!analysis!of!the!ubi1∆ub2HA!ubi2∆!strain!reveals,!in!addition!to!the!appearance!of! halfAmer!polysomes,!a!clear!decrease!in!normal!polysomes!(Figure(5B).!These!profiles! resemble!those! obtained! upon! the! depletion!of! eL40,! including!the! nearly! invariant! free!60S/40S!ratio! found! (FernándezAPevida! et! al.,! 2012),! therefore,! confirming! that! the!amount!of!eL40!is!indeed!limited!in!this!strain.!Consistently,!amplification!of!the! copy!number!of!the!ubi1∆ub!allele!by!positioning!it!in!a!CEN!plasmid,!lead!to!wildAtype! FIGURE(4.(Analysis(of(the(eL40A]HA(protein(levels(in(the(different(mutants(of(this(study .!WildAtype! and! the! indicated! strains! expressing! distinct! CAterminally! HAAtagged! Ubi1! constructs! from! the! genomic! locus! or! from! a! centromeric! plasmid,! in! a! wild! type! UBI2!or! a! ubi2∆!null! mutant! background,! were! grown! in! liquid! YPD! medium! at! 30! ºC.! Total! cell! extracts !were! prepared! and! subjected!to!Western!analysis!using!antiAHA!and!antiAPgk1!(loading!control)!antibodies.! The!mature! eL40AAHA!and! the!Smt3AeL40AAHA! precursor!are!indicated.!The!relative! intensities! of! the! specific! bands!from!these!western!blots!were!quantified,!and!the!ratios!of!the!relative!intensities!between! eL40AAHA!and!the!corresponding!Pgk1!signal!were!calculated.!The!ratios!obtained!for!the!UBI1AHA! strain,!which!were!arbitrarily!set!to!1.0,!were!used!for!normalisation.( ! ! 59( Chapter 1 polysome!profile,!as!this!overcomes!the!limitation!in!the!eL40!accumulation!(Figure(4( and( 5C).!Altogether,!these!results!indicate!that!the!deletion!of!the!ubiquitin!moiety! from! a! genomically! expressed! ubiquitinAfree! Ubi1∆Ub! precursor,! in! the! absence! of! eL40B,! leads! to! a! decrease! production! of! eL40A! and! as! a! consequence! of! 60S! rA subunits.!Thus,!we!conclude!that,!as!also!occurring!for!the!ubiquitin!moiety!of!Ubi3! (Finley!et!al.,!1989;!Lacombe!et!al.,!2009),!the!ubiquitin!moiety!of!Ubi1!is!important! but!not!strictly!required!for!ribosome!synthesis.!! ! We!also!assess!the!effects!of!the!replacement!of!the!ubiquitin!by!a!SUMO!moiety! on! ribosome! biogenesis.! As! shown! in! Figure( 5D,! the! SMT32S2eL40A2HA#ubi2∆# strain! displayed!a!polysome!profile!showing!an!increase!in!the!amount!of!free!60S!rAsubunits! vs!40S! rAsubunits! as! well! as! halfAmer! polysomes.! This! profile! is! typical! of! mutants! partially!impaired!in!60S!to!40S!rAsubunit!joining!(e.g.!(DeLabre!et!al.,!2002;!Eisinger!et! al.,!1997;!FernándezAPevida!et!al.,!2012))!that,!strikingly,!we!have!also!observed!for! other!ubi1!mutants!unable!to!efficiently!remove!the!ubiquitin!moiety!of!Ubi1!(MartínA Villanueva!et!al.,!2019).! FIGURE(5.(The(genomically(integrated(ubi1∆ub6HA+and+SMT36eL40A( alleles(lead(to(a(deficit(in(60S( r]subunits.( Strains(SMY256! (UBI12HA! ubi2∆)! (A),! SMY257! (ubi1∆ub2HA! ubi2∆)! (B),! SMY215! [YCplac111Aubi1∆ub2HA]!(ubi1∆!ubi2∆#+#ubi1∆ub2HA)!(C),!and!SMY258!(SMT3AeL40AAHA!ubi2∆)! (D)! were!grown!in!YPD!medium!at!30ºC!to!an!OD600! of!about!0.8.!Cell!extracts!were!prepared!and!10!A260! units! of! each! extract! were! resolved! in! 7–50%! sucrose! gradients.! The! A245! was! continuously! measured.!Sedimentation!is!from!left!to!right.!The!peaks!of!free!40S!and!60S!rAsubunits,!80S!vacant! ribosomes/monosomes!and!polysomes!are!indicated.!HalfAmer!polysomes!are!labeled!by!arrows.!( ! ! 60( Chapter 1 We! next! studied,! in! the! different! mutants,! the! distribution! of! their! respective! eL40AAHA! rAprotein! variants! in! translating! ribosomes! by! fractionation! analysis.! No! apparent! differences! were! found! between! the! distribution! of! eL40AHA! expressed! either! from! the! ubi1∆ubAHA! or! the! UBI1AHA! alleles.! In! both! cases,! eL40AAHA! is! detected!in!80S!ribosome!and!polysome!fractions!as!also!is!the!60S!rAsubunit!control! protein! uL3.! This! result! indicates! that! the! ubiquitin! moiety! is! not! required! for! the! assembly!of!eL40A!protein!into!functional!60S!rAsubunits!(Figure(6).!In!contrast,!when! the!SMT32S2eL40A2HA#ubi2∆#strain!was!studied,!while!mature!eL40AAHA!fractionated! similarly!as!the!60S!rAsubunit!control!protein!uL3,!the!nonAcleaved!Smt3ASAeL40AAHA! mainly!peaked!in!the!60S!and!to!a!lesser!extend!in!the!80S!fractions!(Figure( 6).!This! FIGURE( 6.( Incorporation( into( translating( ribosomes( of( the( eL40A]HA( protein( derived( of( the( Ubi1∆ub]HA( and( Smt3]eL40A]HA( precursors.( Strains!SMY256!(UBI1AHA!ubi2∆),!SMY257!(ubi1∆ubA HA!ubi2∆)!and! SMY258! (SMT32eL40AAHA!ubi2∆)!were!grown!in! YPD!medium!at!30! ºC! to! midAlog! phase.!Cell!extracts!were!prepared!and!10!A260! units!of!each!extract!were!resolved!in!7–50%!sucrose! gradients.!Fractions!were!collected!from!the!gradients,!then!the!proteins!were!extracted!from!each! fraction,!and!equal!volumes!analyzed!by!western!blotting!using!antiAHA!and!antiAuL3!antibodies.!The! position!of!free!40S!and!60S!rAsubunits,!80S!vacant!ribosomes/monosomes!and!polysomes,!obtained! from!the!recorded!A245!profiles,!are!shown.!T,!total!cell!extract.!( ! ! 61( Chapter 1 precursor!protein!is!basically!absent!from!the!polysomal!fraction.!This!result,!which!is! similar!to!that!we!have!previously!observed!for!nonAcleaved!Ubi1!precursor!variants! (MartínAVillanueva! et! al.,! 2019),! indicates! that! the! Smt3ASAeL40AAHA! precursor! can! assemble! into! preA60S! rAparticles! that! are! unable! to! engage! in! subunit! joining! and! possible! also! in! translation! elongation.! Strikingly,! uL3! but! not! mature! eL40A! was! identified!in!the!60S!peak!(see!Figure(6,!bottom!panel,!lanes!7A10);!this!result!further! supports! the! hypothesis! that! most! of! the! 60S! rAparticles! containing! eL40A! are! efficiently!recruited!into!polysomes!while!those!containing!Smt3ASAeL40AAHA!are!not! and!accumulate!in!this!area!of!the!gradient!(see!profile!in!Figure(5D).! ! Expression(in+trans(of(free(ubiquitin(fails(to(rescue(the(deficiencies(of(the(ubi1∆ub]HA( ubi2∆(and(SMT36S6eL40A]HA(ubi2∆(mutants( It!could!be!reasoned!that!the!mutants!used!in!this!study,!ubi1∆ub2HA!ubi2∆!and! SMT32S2eL40A2HA#ubi2∆,#may!contain!decreased!levels!of!total!cellular!free!ubiquitin! since,! in! principle,! this! molecule! could! only! be! supply! by! the! UBI3! gene! in! actively! growing!mutant!cells.!Thus,!the!defects!we!observed!in!the!above!mutants!might!be! rather!due!to!an!ubiquitin!shortage.!In!this!scenario,!ectopic!in#trans!expression!of!free! ubiquitin!would!result!in!a!suppression!of!the!negative!phenotypes!of!these!mutants.! To! directly! test! this! possibility,! we! transformed! the! different! ubi1! strains! with! a! plasmid!expressing!the!ubiquitin!moiety!to!supplement!the!available!amount!of!free! ubiquitin! or! with! an! empty! control! plasmid.! As! shown! in! Figure( 7A,! the! growth! phenotype! of! the! UBI1AHA! ubi2∆,# ubi1∆ubAHA! ubi2∆! and! SMT32S2eL40A2HA! ubi2∆! strains! did! not! change! when! in# trans!expressing! free! ubiquitin! from! a! plasmid! compared!to!the!control.!In!addition,!we!performed!polysome!profile!analyses!in!the! different!ubi1!mutants!transformed!with!either!an!empty!control!plasmid!or!a!plasmid! expressing!the!ubiquitin!moiety.!As!a!result,!the!expression!of!free!ubiquitin!did!not! restore!the!60S!rAsubunit!shortage!observed!for!the!ubi1∆ubAHA!ubi2∆!and!SMT32S2 eL40A2HA!ubi2∆!mutants!nor!change!the!profiles!obtained!for!the!isogenic!UBI1AHA! ubi2∆#strain!(Figure( 7B!and!data!not!shown).!Altogether,!these!results!indicated!that! the! contribution! of! ubiquitin! to! the! synthesis! and! assembly! of! eL40! is! exclusively! exerted!in#cis!not!been!able!to!perform!it!when!expressed!in#trans!as!a!free!molecule.!! ! ! 62( Chapter 1 ( FIGURE( 7.( Expression( of( free( ubiquitin( in+ trans(does( not( restore( the( deficiencies( conferred( by( neither(the( ubi1∆ub6HA(ubi2∆+nor(the(SMT3]eL40A]HA(ubi2∆+alleles.((A)!Growth!comparison!of!the! strains! W303A1A! (Wild! type),! SMY256! (UBI12HA!ubi2∆),!SMY257!(ubi1∆ub2HA! ubi2∆)!and! SMY258! (SMT3AeL40AAHA!ubi2∆) !transformed!with!either!an!empty!YCplac111!(vector)!or!YCplac111AUb!(Ub)! plasmids.!Transformants!were!selected!on!SDALeu!plates!and!then!spotted!in!fivefold!serial!dilution! steps!onto!SDALeu!plates,!which!were!incubated!for!4!days!at!22!ºC!and!for!3!days!at!30!ºC!and!37!ºC.! (B)!Polysome!profile!analysis!of!ubi1∆ub2HA!ubi2∆!cells!harboring!an!empty! YCplac111!(vector)!or! YCplac111AUb!(Ub)! plasmids.! Cells! were! grown! in! SDALeu! at! 30ºC! to! an!OD600! of! about! 0.8.! Cell! extracts!were!prepared!and!10!A260!units!of!each!extract!were!resolved!in!7–50%!sucrose!gradients.! The!A245! was!continuously!measured.!Sedimentation!is!from!left!to!right.!The!peaks!of!free!40S!and! 60S!rAsubunits,!80S!vacant!ribosomes/monosomes!and!polysomes!are!indicated.!HalfAmer!polysomes! are!labeled!by!arrows.( ! ! 69( Chapter 1 et!al.,!2016),!however,!this!complex!has!not!been!shown!to!facilitate!eL40!assembly.! (ii)! A! more! plausible! scenario! is! that! suggesting! that! ubiquitin! has! a! cisAacting! molecular!chaperone!role!to!facilitate!translation!and!folding!of!eS31!and!eL40.!Only!a! properly!folded!rAprotein!could!assemble!while!the!misfolded!fraction!will!aggregate! and!degrade.!In!agreement!with!this!cisAacting!role!of!ubiquitin,!we!showed!that!the! genomic!expression!of!ubiquitinAfree!eL40A!was!clearly!reduced!(Figure(4)!and!that!the! replacement!of!the!ubiquitin!moiety!of!Ubi1!by!the!ubiquitinAlike!protein!SUMO!(yeast! Smt3),!which!is!known!to!increase!the!efficient!expression!of!fused!proteins!(Baker,! 1996;! Butt! et! al.,! 1989;! Ecker! et! al.,! 1989;! Marblestone! et! al.,! 2006),! was! able! to! partially! suppress! the! ubi1∆ubAHA#ubi2∆! mutant,! specially! at! high! temperatures! (Figure( 3)! and! lead! to! better! eL40AAHA! protein! yields! (Figure( 4).! The! fact! that! the! Smt3ASAeL40AAHA!precursor!is!not!efficiently!processed!(Figure(4(and(6)!could!be!the! reason! for! just! a! mildly! suppression,! as! ubiquitin! release! from! eL40! is! required! for! cytoplasmic!maturation!and!function!of!mature!ribosomal!subunits!(MartínAVillanueva! et!al.,!2019)!(see!also!Figure(6).!To!further!explore!the!role!of!ubiquitin!as!a!chaperone! of!rAproteins!eL40!and!eS31,!we!have!analyzed!the!aggregation!status!of!different!eL40! and! eS31! protein! variants.! In! general,! most! rAproteins! have! a! high! tendency! to! aggregate!during!or!after!their!synthesis!due!to!their!particular!characteristics,!such!as! its!extremely!high!isoelectric!point!and!the!presence!of! unstructured! or! intrinsically! disordered!extensions!(Jäkel!et!al.,!2002).!These!features!have!permitted!the!natural! selection!of!proteins!known!as!dedicated!chaperones!and!escortins,!which!help!import! and/or! assembly! of! individual! rAproteins,! thus! impeding! their! degradation,! inappropriate! interaction! with! other! cellular! components! and! aggregation! (EspinarA Marchena!et!al.,!2017;!Peña!et!al.,!2017;!Pillet!et!al.,!2017).!Additionally,!rAproteins!are! among!the!major!client!proteins!of!the!general!ribosomeAassociated!chaperones!such! as! the! nascent! polypeptideAassociated! complex! (NAC)! and! the! SsbAribosomeA associated!complex!(SsbARAC)!(Koplin!et!al.,!2010).!It!has!been!shown!that!the!loss!of! NAC! and! SsbARAC! complexes! causes! aggregation! of! most! rAproteins! from! both! rA subunits!(Koplin!et!al.,!2010)!(Figures(8(and(9).!Although!originally!rAproteins!eL40!and! eS31!were!not!found!among!the!species!identified!in!the!aggregation!analysis!of!cells! lacking!NAC!and!SsbARAC,!our!results!show!that,!at!least,!the!ubiquitin!moiety!fused!to! eS31! does! not! prevent! its! specific! aggregation! in! the! absence! of! these! chaperone! ! ! 70( Chapter 1 complexes! (Figure( 9).! Interestingly,! the! ubiquitin! deletion! from! Ubi3! caused! a! mild! enhancement! of! the! aggregation! levels! of! eS31.! However,! this! enhancement! is! not! only!specific!for!eS31!but!also!for!the!rest!of!rAproteins!tested!(uL3,!uL29!and!uS3,!see! Figure( 9).!Indeed,!we!show!herein!that!a!defect!in!ribosome!biogenesis!(e.g.!ubi3∆,! rps12∆,!rpl39∆!or!dob121!mutants)!leads!to!a!substantial!increase!on!the!amount!of! insoluble! material! detected! in! polyacrylamide! gels,! with! aggregation! of! rAparticles,! similarly! to! that! observed! in! cells! lacking! NAC! and! SsbARAC! (Figure( 8( and( 9).! Very! recently,!Tye!et#al.!demonstrated!that!an!imbalance!in!the!synthesis!of!rAproteins!and! rRNAs!leads!to!the!rapid!aggregation!of!newly!synthetized!rAproteins!(Tye!et!al.,!2019).! Thus,!it!is!likely!that!the!modest!increase!in!the!aggregation!status!detected!in!ubi3∆ub! cells!could!be!attributable!to!the!ribosome!biogenesis!defect!caused!by!the!ubi3∆ub! mutation,!which!impair!40S!rAsubunit!synthesis!(Lacombe!et!al.,!2009).!Similarly,!the! strain! UBI1AHA! ubi2∆! showed! a! substantial! amount! of! insoluble! material! and! aggregation!of!rAproteins,!as!both!the!ubi2∆!mutation!and!to!the!lesser!extend!the!HAA tag!of!UBI1!interfere!with!cell!growth!and!ribosome!biogenesis!(FernándezAPevida!et! al.,!2012;!Kruiswijk!et!al.,!1978;!Lacombe!et!al.,!2009;!MartínAVillanueva!et!al.,!2019)! (Figure( 2).! Unfortunately,! this! basal! status! seems! to! be! high! enough! to! mask! the! possible!additional!negative!effect!caused!by!the!ubi1∆ub!allele.!In!contrast,!the!fusion! of!Smt3!to!eL40!seems!to!modestly!ameliorate!the!aggregation!levels!of!this!rAprotein.! Altogether,!these!experiments,!although!not!conclusive,!strongly!suggest!that!ubiquitin! could!be!assisting!in#cis!the!folding!of!their!respective!fused!rAproteins.! ( ! ! ( ( ( ! ! 71( Chapter 1 MATERIALS(AND(METHODS( ( Strains(and(microbiological(methods( All!yeast!strains!used!in!this!study!are!listed!in!Table(1!and!are!derived!from!the! diploid!W303!strain!background!(Thomas!and!Rothstein,!1989).! ! A!twoAstep!allele!replacement!method!was!used!to!replace!genomic!wildAtype! UBI1!by!UBI1AHA,!ubi1∆ubAHA!or!SMT32eL40AHA!alleles!(Klockner!et!al.,!2009).!Briefly,! SMY106,!which!is!an!ubi1::klURA3!(URA3!gene!from!Kluyveromyces#lactis)!haploid!null! mutant!strain,!was!coAtransformed!with!100!ng!of!empty!YCplac111!vector!and!1!µg!of! DNA!fragments!containing!the!UBI1AHA,!ubi1∆ubAHA,!or!SMT3AeL40AHA!alleles!excised! by! EcoRI/HindIII! digestion! from! YCplac111AUBI1AHA,! YCplac111Aubi1∆ubAHA,! or! YCplac111ASMT3AeL40AHA,! respectively.! Transformants! were! selected! onto! SDALeu! plates.!Then,!several!clones!were!replicaAplated!onto!5AFOAAcontaining!plates!to!select! for! those! that! had! lost! the!klURA3! marker! because! of! a! siteAspecific! recombination! event.!Candidates!were!analysed!by!colony!PCR!and!sequencing.!! ! Strains!SMY215!and!SMY216!are!generated!by!transforming!the!UBI1/2!shuffle! strain! (TAY001)! with! YCplac111Aubi1∆ubAHA! or! YCplac111ASMT3AeL40AHA! plasmids;! transformants!were!restreaked!on!5AFOAAcontaining!plates! to!counterAselect!against! the!presence!of!pHT4467∆AUBI1.! ! Yeast!cells!were!grown!at!the!indicated!temperatures!either!in!YPD!medium!(1%! yeast!extract,!2%!peptone!and!2%!glucose)!or!SD!medium!(synthetic!dextrose;!0.15%! yeast!nitrogen!base,!0.5%!ammonium!sulphate)! supplemented! with!the!appropriate! amino! acids! and! bases! as! nutritional! requirements,! and! containing! 2%! glucose! as! carbon!source.!To!prepare!plates,!2%!agar!was!added!to!the!media!before!sterilization.! Yeast!genetic!techniques!and!growth!media!have!been!previously!described!(Burke!et! al.,!2000).!Yeast!cells!were!transformed!by!the!lithium!acetate!method!(Gietz!et!al.,! 1992).!For!tetrad!dissection,!a!Singer!MSM!400!micromanipulator!was!used.!Standard! molecular! biology! techniques! were! carried! out! according! to! the! Sambrook! ! ! 72( Chapter 1 specifications!(Sambrook!et!al.,!1989).!Escherichia#coli!DH5α!was!used!for!cloning!and! propagation!of!plasmids!(Sambrook!et!al.,!1989).! ! Plasmids( Plasmids!used!in!this!study!are!listed!in!Table( 2.!Cloned!DNA!fragments!were! generated!by!a!fusion!PCR!strategy!to!include!a!single!HA!epitope!at!the!CAterminal! end!of!eL40A!in!wildAtype!and!mutant!versions!of!UBI1.!All!constructs!were!verified!by! DNA! sequencing.! Description! of! the! oligonucleotides! used! for! the! PCRs! and! information! on! the! construction! of! the! different! plasmids! will! be! available! upon! request.! ! Polysome(analysis(and(sucrose(gradient(fractionation( Cell! extracts! for! polysome! analysis! were! prepared! and! analysed! as! previously! described!(Foiani!et!al.,!1991;!Kressler!et!al.,!1997).!Ten!A260!units!of!each!cell!extract! were! loaded! onto! 7A50%! sucrose! gradients.! These! gradients! were! centrifuged! at! 39,000!rpm!in!a!Beckman!Coulter!rotor!SW41!for!2!h!45!min;!the!A254!was!continuously! monitored! using! an! ISCO! UAA6! system.! When! needed,! fractions! of! 0.5! ml! were! collected!and!proteins!were!precipitated!from!each!fraction!using!trichloroacetic!acid! as! previously! described! (de! la! Cruz! et! al.,! 1998a).! The! precipitated! fractions! were! resuspended! in! 2X! Laemmli! loading! buffer;! an! equal! volume! of! each! fraction! was! separated!by!SDSAPAGE!and!analysed!by!western!blotting.!! ! Western(blot(analysis(and(antibodies( Total!yeast!protein!extracts!were!prepared!by!the!alkaline!lysis!method!of!Yaffe! and!Schatz!(Yaffe!and!Schatz,!1984),!which!immediately!"freezes"!the!in#vivo!protein! content,!thus,!preventing!rapid!protein!turnover!reactions.!Proteins!were!separated!by! SDSAPAGE! and! analysed! by! western! blotting! according! to! standard! procedures! (Sambrook! et! al.,! 1989).! The! following! primary! antibodies! were! used:! mouse! monoclonal! antiAHA! 16B12! (Covance),! antiAPgk1! (Invitrogen)! and! antiAuL3! (gift! from! J.R.! Warner)! (Vilardell! and! Warner,! 1997);! rabbit! polyclonal! antiAuL29! (gift! from! M.! Seedorf)!(Frey!et!al.,!2001)!and!antiAuS3!(gift!from!M.!Seedorf)!(Frey!et!al.,!2001).!Goat! antiAmouse! or! antiArabbit! horseradish! peroxidaseAconjugated! antibodies! (BioARad)! ! ! 73( Chapter 1 were! used! as! secondary! antibodies.! Immune! complexes! were! visualised! using! a! chemiluminescence!detection!kit!(SuperASignal!West!Pico,!Pierce)!and!a!ChemiDocTM! MP!imaging!system!(BioARad).! ! Analysis(of(aggregated(proteins( Isolation! of! aggregated! proteins! was! done! from! whole! cell! extracts! as! exactly! described! by!Koplin! et# at.!(Koplin! et! al.,! 2010)! but! following! the! sonication! modifications!described!in!Panasenko!et#al.!(Panasenko!and!Collart,!2012).!Samples!of! whole! cell! extracts! and! aggregated! protein! were! resuspended! in! Laemmli! sample! buffer,!separated!by!SDSAPAGE!and!analysed!by!colloidal!blue!Coomassie!staining!and! western!blotting.!! ! ! ! ! 74( Chapter 1 ! Table(1.(Yeast(strains(used(in(this(study.( ( Strain((a)( Relevant(genotype( Source( ! W303A1A! # MATa(ade221#his3211,15#leu223,112#trp121#ura321! ! (Thomas! and! Rothstein,! 1989)! W303A1B! As!W303A1A!but!MATα! (Thomas! and! Rothstein,! 1989)! TAY001! MATa!ubi1::kanMX4!ubi2::kanMX4!ade3::kanMX4![pHT4467∆AUBI1]! (MartínA Villanueva! et! al.,!2019)! SMY106! MATa!ubi1::klURA3! This!study! SMY113! MATa!UBI1AHA! This!study! SMY256! MATa!UBI1AHA!ubi2::kanMX4! This!study! SMY107! MATa!ubi1∆ubAHA! This!study! SMY257! MATα!ubi1∆ubAHA!ubi2::kanMX4! This!study! SMY215! MATa!ubi1::kanMX4! ubi2::kanMX4! ade3::kanMX4! [YCplac111A ubi1∆ubAHA]! This!study! JDY922! MATa!ubi2::kanMX4! (FernándezA Pevida! et! al.,! 2012)! SMY218! MATα!SMT3AeL40AAHA! This!study! SMY258! MATα!SMT3AeL40AAHA!ubi2::kanMX4! This!study! SMY216! MATa!ubi1::kanMX4!ubi2::kanMX4!ade3::kanMX4![YCplac111ASMT3A eL40AAHA]! This!study! JDY532! MATa!ssb1::HIS3MX6!ssb2::natNT2! This!study! SMY324! MATa!ssb1::HIS3MX6!ssb2::natNT2!UBI3AHA::kanMX4! This!study! TLY56.D3!! MATa!UBI3AHA::kanMX4! (Lacombe! et! al.,!2009)! TLY61.A2!! MATa!ubi3∆ubAHA::kanMX4! (Lacombe! et! al.,!2009)! TLY14.3C! MATα!ubi3::HIS3MX6! (Lacombe! et! al.,!2009)! SMY315! MATα!rps12::kanMX4! This!study! ORY211! MATα!rpl39::natNT2! This!study! MS157A1A!! ! MATα!dob121! (de! la! Cruz! et! al.,!1998b)! ! (a)Strains!used!in!this!study!are!isogenic!with!W303.!To!simplify,!only!the!corresponding!mating! type!of!the!W303!relevant!genotype!is!provided.! ! ! ! ! 75( Chapter 1 ! Table(2.(Plasmids(used(in(this(study( ( Name((collection(name)( Relevant(information( Reference( ! pHT4467∆AUBI1! ! CEN6!(instable),! URA3,! ADE3.! WildAtype! Ubi1;! promoter!and!terminator!of!UBI1! ! (MartínA Villanueva! et! al.,! 2019)! YCplac111AUBI1AHA! (pDK4131)! CEN,! LEU2.! CAterminally! 1xHAAtagged! eL40A;! promoter!and!terminator!of!UBI1! (MartínA Villanueva! et! al.,! 2019)! YCplac111Aubi1∆ubAHA! (pDK4192)! CEN,! LEU2.! Allele! ubi1∆ubAHA! (MAII);! promoter! and!terminator!of!UBI1! This!study! YCplac111ASMT3AeL40AHA! (pDK4193)! CEN,! LEU2.! Allele! SMT3AeL40AAHA! (GGASAII);! promoter!and!terminator!of!UBI1! This!study! YCplac111! CEN,!LEU2!! (Gietz! and! Sugino,! 1988)! YCplac111AUb! ! CEN,!LEU2.!Ubiquitin! This!study! ! ! ! ! ! CHAPTER(2( ( Role(of(the(beak(ribosomal(protein(eS12(in(ribosome( biogenesis(and(function(in(Saccharomyces+cerevisiae ! ! 85( Chapter 2 Loss(of(eS12(leads(to(a(strong(40S(r]subunit(shortage! To!study!the!contribution!of!yeast!eS12!to!ribosome!biogenesis,!we!first!analysed! polysome!profiles!from!cell!extracts!of!the!rps12!deletion!mutant!and!compared!it!to! those!of!isogenic!ubi3∆!and!wildAtype!strains.!As!shown!in!Figure(3A,!polysome!profile! analysis!of!the!rps12∆!mutant!revealed!a!clear!reduction!in!free!40S!rAsubunits!and!a! huge! increase! in! free! 60S! rAsubunits;! the! polysomal! content! was! also! drastically! reduced.!The! profile!was! similar! to!those! of! the! single! ubi3∆!and! the! double! ubi3∆# rps12∆!isogenic!mutants!(Figure( 3A).!Thus,!the!rps12∆!alone!or!in!combination!with! the!ubi3∆!mutation!lead!to!a!shortage!of!40!S!rAsubunits.!In!agreement,!quantification! of!total!rAsubunits!by!lowAMg2+!sucrose!gradients!showed!that!deletion!of!either!eS12! or!eS31!leads!to!a!ca.!55!%!reduction!of!the!40S!to!60S!rAsubunits!compared!to!the! wildAtype! control.! This! reduction! was! of! about! 70%! for! the! double! rps12∆# ubi3∆! mutant!(Figure(3B).!We!conclude!that!the!absence!of!eS12!rAprotein!results!in!a!severe! deficiency!in!40S!rAsubunits!relative!to!60S!rAsubunits,!which!is!more!pronounced!when! eS31!rAprotein!is!simultaneously!lacking.! ! Deletion(of(RPS12(impairs(rRNA(processing( To!determine!whether!the!rps12∆!mutant!was!defective!in!the!formation!rather! than!the!stability!of!40S!rAsubunits,!we!examined!the!kinetics!of!rRNA!production!by! pulseAchase!analysis!with!the!rps12∆!strain!and!its!isogenic!wildAtype!counterpart.!Both! strains!were!first!transformed!with!an!empty!YCplac33!plasmid!(CEN,!URA3,!Table(2)!to! make!them!prototrophic!for!uracil.!Then,!they!were!grown!in!liquid!SDAUra!medium!to! midAlog!phase!at!30!ºC.!The!cells!were!pulseAlabelled!for!2!min!with![5,6A3H]uracil,!then!! chased!for!5,!15,!30!and!60!min!with!a!large!excess!of!nonAradioactive!uracil.!Total!RNA! was! extracted! from! each! sample! and! analysed! by! agarose! and! acrylamide! electrophoresis,!followed!by!transfer!to!nylon!membranes!and! exposition! to!tritium! sensitive!phosphor!screens;!results!were!revealed!in!a!phosphor!imaging!system.!As! shown!in!Figure(4A,!in!wildAtype!cells,!the!early!preArRNA!precursors!were!converted! rapidly!into!27S!and!20S!species,!which!were!further!processed!into!mature!25S!and! 18S!rRNA,!respectively.!After!15!min!of!chase,!practically!all!label!was!in!the!mature! rRNAs.!Compared!with!the!wildAtype!control,!processing!of!27S!preArRNAs!was!delayed! in!the!rps12∆!mutant!since!these!precursors!could!be!detected!after!the!30!min!chase!! ! ! 86( Chapter 2 FIGURE(3.(Deletion(of(RPS12(results(in(a(deficit(in(40S(r]subunits.((A)!Polysome!profile!analysis!of! W303A1A! (wild! type),! SMY315! (rps12∆),! TLY14.3C! (ubi3∆)! and! the! double! mutant! strain! SMY372! (rps12∆#ubi3∆)!strains.! The!peaks!of!free!40S!and!60S!rAsubunits,!80S!free!couples/monosomes!and! polysomes! are! indicated.! (B)! Quantification! of! the! 40S! rAsubunit! relative! to! the! 60S! rAsubunit! abundance!in! the! strains! described! above.! Cell! extracts! were! prepared! under! polysome! runAoff! conditions,!by!the!omission!of!cycloheximide,!in!a!buffer!lacking!MgCl2!to!dissociate!ribosomes!in!the! 40S!and!60S!rAsubunits.! The!peaks!of!total!40S!and!60S!rAsubunits!are!indicated.!The!area!under!the! curve!was!determined!for!individual!peak,!and!the!60S/40S!ratios!was!calculated!for!each!profile;! data!are!the!average! of! three!independent!experiments;! the! relative! standard! deviation!was! less! than!5%!in!each!case. (In!both!experiments,!cells!were!grown!in!YPD!medium!at!30!ºC!to!an!OD600!of! around! 0.8.! Ten! A260!units! of! each! extract! were! resolved! in! 7A50%! sucrose! gradients.! After! centrifugation,! changes! in! the! A254!value! of! each! gradient! were!recorder! by! an! onAline! spectrophotometer.!Sedimentation!is!from!left!to!right.!( ! ! ! 87( Chapter 2 time!point;!consistently,!practically!no!labelled!mature!25S!rRNA!was!detected!at!early! 5!min!or!15!min!chase!time!points!(Figure( 4A).!Similarly,!a!delayed!conversion!of!7S! preArRNAs! into! mature! 5.8S! rRNAs! was! detected! (Figure( 4B).! More! significant,! synthesis!of!18S!rRNA!was!strongly!inhibited!since!20S!preArRNA!persisted!even!after! the!60!min!of!chase.!! ! To!investigate!in!more!detail!the!preArRNA!processing!defects!occurring!in!the! absence! of! eS12,! we! analysed! the! steadyAstate! levels! of! preA! and! mature! rRNAs! by! northern!hybridization!in!the!rps12∆!mutant!and!compared!them!to!those!detected!for! a!ubi3∆!and!a!double!rps12∆#ubi3∆!strains.!As!shown!in!Figure(5,!the!single!rps12∆!and! ubi3∆! strains! and! the! double! rps12∆# ubi3∆! mutants! led! to! practically! identical! preA rRNA!processing!defects.!Thus,!none!of!these!strains!showed!significant!differences!in! the!levels!of!35S!and!32S!preArRNAs!compared!to!the!wildAtype!strain.!However,!some! delay!in!the!early!preArRNA!processing!reactions!might!occur!as!an!aberrant!21S!preA rRNA,! which! extends! from! site! A1! to! A3,! slightly! accumulated! in! the! three! mutant! strains.! Moreover,! the! 27S! and! 7S! preArRNAs! clearly! decreased,! however,! levels! of! mature!25S! and!5.8S! rRNAs! only! slightly! decreased.! In! agreement!with! the!delayed! conversion! of! 20S! preArRNA! to! 18S! rRNA! seen! in! pulse! chase! analysis,! steadyAstate! levels!of! 20S!preArRNA!visibly!accumulated!in!a!similar!manner!in!the!three!mutant! strains!(Figure(5);!as!a!consequence,!levels!of!mature!18S!rRNA!significantly!decreased.! Altogether,!our!results!indicate!that!the!absence!of!eS12!leads!to!a!general!delay!in! preArRNA!maturation!that!is!much!more!pronounced!for!the!20S!preArRNA!conversion! into!mature!18S!rRNA,!which!likely!accounts!for!the!deficit!in!40S!rAsubunit!detected!in! rps12∆!cells.!These!preARNA!processing!defects!are!quite!similar!to!those!observed!in! the!ubi3∆!strain!and!only!slightly!enhanced!by!the!ubi3∆!allele.! ! ! 88( Chapter 2 ! ! ( FIGURE(4.!Deletion(of(RPS12(lead(to(delayed(conversion(of(the(20S(pre]rRNA(to(mature(18S(rRNA.! The!strains!W303A1A!(Wild#type)!and!SMY315!(rps12∆)!were!transformed!with!the!YCplac33!plasmid! and!then!grown!in!SDAUra!to!an!OD600!of!around!0.8!at!30!ºC.!Cells!were!pulseAlabelled!for!2!min!with! [5,6A3H]uracil!and!then!chased!for!5,!15,!30!and!60!min!with!an!excess!of!unlabelled!uracil.!Total!RNA! was! extracted! and! 3,000! cpm! per! sample! were! loaded! and! separated! on! (A)( a! 1.2%! agaroseA formaldehyde! gel! or! (B)( a! 7%! polyacrylamideA8! M! urea! gel,! transferred! to! nylon! membranes,! exposed! to! a! tritium! sensitive! phosphor! screen! and! analyse! by! phosphorimager! scanning.! The! position!of!the!different!preArRNAs!and!mature!rRNAs!are!indicated.( ! 89( Chapter 2 ( ( The(rps12∆(deletion(mutant(accumulates(the(20S(pre]rRNA(in(the(cytoplasm( Nuclear!preA40S!rAparticles!are!rapidly!released!through!the!nucleopore!to!the! cytoplasm,!where!processing!of!20S!preArRNA!to!mature!18S!rRNA!occurs!(Trapman! and! Planta,! 1976;! Udem! and! Warner,! 1973).! Therefore,! a! defect! in! 20S! preArRNA! processing!might!result!from!either!reduced!nuclear!export!of!preA40S!rAparticles!or! impaired!cleavage!of!cytoplasmic!20S!preArRNA.!To!assess!this!issue,!we!first!analysed!! FIGURE( 5.(Absence( of( eS12( leads( to( robust( accumulation( of( 20S( pre]rRNA( and( concomitant( reduction(in(the(steady]state(levels(of(mature(18S(rRNA.! The!strains!W303A1A!(Wild#type),!SMY315! (rps12∆),!TLY14.3C!(ubi3∆)!and!SMY372!(rps12∆#ubi3∆)!were!grown!in!YPD!medium!at!30!ºC!to!an! OD600!of!around!0.8.!Total!RNA!was!extracted!from!each!strain;!equal!amounts!of!RNA!(5!µg)!were! analysed! by! northern! blot.! (A)!Northern! hybridisation! of! highAmolecularAmass! preA! and! mature! rRNAs,!(B)( Northern!blot!analysis!of!lowAmolecularAmass!preA!and!mature!rRNAs.!Signal!intensities! were!measured!by!phosphorimager!scanning!(indicated!below!each!panel)!and!normalised!to!those! obtained! for! the! wildAtype,! arbitrarily! set! to! 1.0.! Probes,! between! parentheses,! are! described! in! Table(3.!(C)!Main!steps!in!preArRNA!processing!are!shown.( ! ! 90( Chapter 2 ! ! FIGURE(6.(The(absence(of(eS12(does(not(impair(export(of(nuclear(pre]40S(ribosomal(particles.((A)( The! strains! W303A1A! (Wild# type),!TLY14.3C! (ubi3∆)! and!SMY315! (rps12∆)!were!transformed!with! plasmids!expressing!the!nucleolar!RFPANop1!reporter!and!either!uL23AeGFP!or!uS3AGFP!from!their! cognate!promoters.!Cells!were!exponentially!grown!in!SDALeu!at!30!ºC!and!then!the!red!and!green! fluorescent! signals! analysed! by! fluorescence! microscopy.! Arrows! indicate! the! position! of! the! nucleolus!in!selected!cells.!(B)! The!localisation!of!eS31!and!eS10!rAproteins!is!not!affected!in!rps12∆! cells.( The!strains!indicated!above!were!transformed!with!plasmids!expressing!GFPAtagged!versions!of! eS10!and!eS31!rAproteins.!Cells!were!exponentially!grown!in!SDALeu!at!30!ºC!and!then,!the!green! fluorescent!signal!analysed!by!fluorescence!microscopy.!Hoechst!staining!reveals!the!nucleoplasm,! whose!position!was!indicated!by!arrows!in!selected!cells.( ! 91( Chapter 2 the!subcellular!localization!of!a!GFPAtagged!version!of!uS3!rAprotein,!which!assembles! in!the!nucleus!(Koch!et!al.,!2012;!Mitterer!et!al.,!2016),!in!wildAtype,!rps12∆!and!ubi3∆! cells.!As!shown!in!Figure(6A,!both!uS3AeGFP!and!the!60S!rAsubunit!reporter!uL23AeGFP,! which!also!assembles!in!the!nucleus!(e.g.!(Hurt!et!al.,!1999)),!were!cytoplasmic!in!the! wildAtype!and!mutant!rps12∆!and!ubi3∆!strains.!We!also!analysed!the!localisation!of! GFPAtagged!versions!of!eS10!and!eS31,!which!are!close!neighbour!of!eS12!at!the!beak! (Rabl!et!al.,!2011);!eS31!has!been!shown!to!assemble!in!the!nucle(ol)us!(FernándezA Pevida!et!al.,!2016;!Sun!et!al.,!2017)!and!eS10!has!been!suggested!to!predominantly! assemble!late!in!the!cytoplasm!(Collins!et!al.,!2018;!Strunk!et!al.,!2011).!As!shown!in! Figure(6B,!no!nuclear!accumulation!of!these!two!reporters!could!either!be!observed!in! the!rps12∆!strain.!! ! We!also!visualised!the!20S!preArRNA!and!its!precursors!(35S,!33S!and!32S!preA rRNAs)!by!fluorescent!in!situ!hybridisation!(FISH)!in!the!rps12∆!and!ubi3∆!strain!using!a! probe!complementary!to!positions!within!the!DAA2!region!of!ITS1.!As!shown!in!Figure(7,! as!expected,!a!mainly!nucleolar!FISH!signal!was!detected!in!wildAtype!cells!given!that! 35S,! 33S! and! 32S! preArRNAs! are! nucleolar! and! cytoplasmic! 20S! preArRNA! is! rapidly! processed!to!mature!18S!rRNA!in!the!cytoplasm.!However,!the!FISH!signal!turned!to!be! also!abundant!in!the!cytoplasm!in!the!rps12∆!and!ubi3∆!strains,!thus,!suggesting!that! 20S!preArRNA!is!efficiently!exported!in!these!mutants.!Altogether,!these!results!reveal! that!preA40S!rAparticles!export!from!the!nucleus!is!neither!blocked!in!the!rps12∆!or! ubi3∆!mutant.!Moreover,!the!increased!levels!of!20S!preArRNA!detected!rps12∆!and! ubi3∆! mutant! cells! are! the! result! of! an! impairment! of! cytoplasmic! 20S! preArRNA! processing.! ! The( accumulated( 20S( pre]rRNA( is( not( efficiently( incorporated( into( translating( ribosomes(in(rps12∆(cells( It! has! been! previously! reported! that,! in! some! mutants,! preA40S! rAparticles! containing! the! 20S! preArRNA! could! be! efficiently! incorporated! into! translating! ribosomes!(Lacombe!et!al.,!2009)!(GarcíaAGómez!et!al.,!2014;!Pertschy!et!al.,!2009).!In! contrast,!in!wildAtype!cells!and!most!mutant!that!accumulate!20S!preArRNA,!preA40S!rA particles!were!only!found!to!peak!together!with!free!40S!rAsubunits!or!80S!ribosomes! ! ! 92( Chapter 2 (e.g.!(Ghalei!et!al.,!2017;!Granneman!et!al.,!2005;!Jakovljevic!et!al.,!2004;!Soudet!et!al.,! 2010;!Strunk!et!al.,!2012)).! ! To! examine! whether! preA40S! rAparticles! could! engage! in! translation! in! rps12∆! mutant!cells,!we!performed!polysome!gradients!in!the!rps12∆!strain!and!subsequently! analyse!the!distribution!of!20S!preArRNA!in!the!different!fractions!of!the!gradients!by! northern!blotting.!The!results!were!compared!to!those!of!an!equivalent!study!done! with! wildAtype! and! ubi3∆! cells.! As! expected,! the! bulk! of! 20S! preArRNA! coAmigrated! basically!with!the!free!40S!peak!in!wildAtype!cells!(Figure(8).!However,!in!rps12∆!and! ubi3∆!cells,! 20S! preArRNA! was! predominantly! found! in! the! 80S! peak,! its! maximum! shifted! one! fraction! left! to! the! maximum! of! 25S/18S! in! this! peak! (Figure( 8).! Some! portion!of!20S!preArRNA!was!also!distributed!in!the!polysomal!fraction!of!the!gradients! in#rps12∆#and!ubi3∆#cells,!as!it!accumulated!at!the!similar!low!levels!of!25S!and!18S! rRNAs! in! these! fractions! due! to! the! fact! that! in! these! mutants! there! is! a! reduced! content!of!polysomes!(see( Figure( 3A);!indeed,!graphs!of!the!signal!intensities!of!20S! FIGURE(7.(The(20S(pre]rRNA(accumulates(in(the(cytoplasm(of(rps12∆(and(ubi3∆(cells.!The!strains! W303A1A!(Wild#type),!TLY14.3C!(ubi3∆)!and!SMY315!(rps12∆)!were!grown!to!midAlog!phase!in!YPD! medium!at!30!ºC.!Cells!were!fixed!with!formaldehyde,!spheroblasted!and!subjected!to!FISH!using!a! Cy3Alabelled!probe!complementary!to!a!sequence!within!the!DAA2!segment!of!ITS1,!which!hybridises! to!the!35S,!33S,!32S!and!20S!preArRNAs!(Table(3).!DAPI!staining!visualises!the!nucleoplasm.( ! 93( Chapter 2 preArRNA!and!25S!and!18S!mature!rRNAs!confirmed!the!similar!distribution!of!these! three!rRNA!species!only!in!the!gradients!corresponding!to!rps12∆!and!ubi3∆!cells,!but! not!in!those!of!isogenic!wildAtype!cells!(Figure(8B).!These!data!suggest!that!preA40S!rA particles! lacking! either! eS12! or! eS31! can! assemble! into! 80S! ribosomes! but! also! inefficiently!engage!in!translation.! ! ( ( FIGURE(8.(The(absence(of(either(eS12(or(eS31(alters(the(sedimentation(behaviour(of(pre]ribosomal( particles( containing( 20S( pre]rRNA.! (A)!The! strains! W303A1A! (Wild# type),! TLY14.3C! (ubi3∆)! and! SMY315!(rps12∆)!were!grown!to!midAlog!phase!in!YPD!medium!at!30!ºC.!Cell!extracts!were!prepared! and!10!A260!units!of!each!extract!were!resolved!in!7A50%!sucrose!gradients!and!fractionated.!RNA! was! extracted! from! each! fraction! and! analysed! by! northern! blotting! using! the! indicated! probes! (between!parentheses;!see!Table(3).!The!position!of!free!40S!and!60S!rAsubunits,!80S!ribosomes!and! polysomes!are!shown.!Percentage!of!20S!preArRNA!signal!intensity!in!each!case!is!represented.!(B)( The! relative! intensities! of!the! hybridisation! signals! of! the! 20S! preArRNA!and!mature! 25S!and!18S! rRNAs!were!determined!for!each!fraction!by!phosphorimager!analysis!and!plotted!as!arbitrary!units! against!the!fraction!number!(20S!preArRNA,!triangles;!25S!rRNA,!squares;!18S!rRNA,!dots).( ! ! 94( Chapter 2 Dynamics(of(the(trans]acting(factors(Enp1(and(Ltv1(during(40S(r]subunit(biogenesis(in( the(rps12∆(and(ubi3∆(mutants( CryoAEM!analyses!have! shown! that!the!transAacting!factors! Enp1!and! Ltv1! are! present! in! cytoplasmic! preA40S! rAparticles! at! a! position! connecting! the! beak! to! the! head!structure!(see(Figure(1B)!(Heuer!et!al.,!2017;!Johnson!et!al.,!2017;!Scaiola!et!al.,! 2018;!Strunk!et!al.,!2011).!Moreover,!the!release!of!these!two!factors!from!cytoplasmic! preA40S!particles!upon!their!phosphorylation!by!the!kinase!Hrr25!(Schäfer!et!al.,!2006)! seems!to!induce!a!conformational!rearrangement!of!the!beak/head!area,!which!allow! the!stable!positioning!of!uS3!(Ghalei!et!al.,!2016;!Mitterer!et!al.,!2016)!and!the!efficient! recruitment! within! the! head! of! eS10,! uS10,! uS14! and! RACK1! (Collins! et! al.,! 2018).! Given! that! Enp1! associates! to! 90S! preAribosomal! particles,! which! apparently! also! contains!eS12!and!eS31!(Sun!et!al.,!2017)!while!Ltv1!binds!later!to!nucleoplasmic!preA 40S!rAparticles!(Moriggi!et!al.,!2015;!Schäfer!et!al.,!2003;!Seiser!et!al.,!2006),!we!found! particularly!pertinent!to!examine!both!the!functional!relationship!between!eS12,!eS31,! Enp1! and! Ltv1! and! the! dynamics! of! association/release! of! Enp1! and! Ltv1! in! the! absence!of!eS12!and!eS31.! ! We!first!evaluate!whether!the!defects!on!growth!caused!by!the!absence!of!eS12! or!eS31!could!be!enhanced!by!the!absence!of!Ltv1!or!the!partial!loss!of!function!of! Enp1.!It!has!been!reported!that!the!ltv1∆!null!allele!leads!to!a!sg!phenotype!for!growth! at!30!ºC!(see(Figure(9A)(and!significantly!impairs!40S!rAsubunit!synthesis!due!to!defects! in!both!20S!preArRNA!maturation!and!export!of!preA40S!rAparticles!from!the!nucleus!to! the!cytoplasm!(Fassio!et!al.,!2010;!Pertschy!et!al.,!2009;!Seiser!et!al.,!2006).!In!turn,!the! enp121! mutation! renders! a! temperature! sensitive! (ts)! growth! phenotype! at! 37! ºC,! which!is!linked!to!a!severe!40S!rAsubunit!deficiency!due!to!both!the!inhibition!of!preA rRNA!processing!at!the!early!A0AA2!cleavage!sites!and!the!block!of!nucleocytoplasmic! 40S!rAsubunit!export!(Chen!et!al.,!2003;!Schäfer!et!al.,!2003);!however,!at!30!ºC,!the! growth! of! enp121! cells! was! apparently! comparable! to! that! of! an! isogenic! wildAtype! strain!(see( Figure( 9B).!As!shown! in! Figure( 9A,!the!ltv1∆!mutation!confers!synthetic! lethality! when! combined! with! either! the! rps12∆! or! the! ubi3∆! alleles.! Moreover,! as! shown!in!Figure(9B,!the!enp121!mutant!when!combined!with!the!rps12∆!allele!or!the! ubi3∆! mutant! were! synthetically! lethal! or! enhanced,! respectively.! These! findings! ! 101( Chapter 2 DISCUSSION( ! In! last! years,! the! knowledge! about! the! contribution! of! rAproteins! to! ribosome! biogenesis! has! increased! significantly! (de! la! Cruz! et! al.,! 2015).! However,! some! rA proteins!remain!uncharacterized!yet.!In!this!work,!we!have!addressed!the!role!of!the! eukaryotic!specific!rAprotein!eS12!in!yeast!40S!rAsubunit!biogenesis!and!function.!The!rA protein!eS12!is!located!on!the!beak!of!the!small!rAsubunit,!closed!to!eukaryotic!specific! rAproteins!eS10!and!eS31.!With!this!study,!we!provide!the!first!report!dissecting!the! contribution!of!the!rAproteins!of!the!beak!to!ribosome!biogenesis.! Analysis! of! the! rps12! null! mutant! shows! that,! contrary! to! most! rAproteins! in! yeast,!eS12!is!not!an!essential!rAprotein,!albeit!absence!of!eS12!strongly!impairs!cell! growth!at!all!tested!temperatures!in!different!genetic!backgrounds!(Figure(2).!Similar! results!have!been!reported!by!(Steffen!et!al.,!2012).!The!absence!of!nonessential!rA proteins!reduces!cell!growth!rates!to!different!extents,!being!almost!dispensable!for! growth!in!some!cases!(Steffen!et!al.,!2012).!! Relative! to! the! wild! type,! deletion! of! RPS12# causes! a! strong! 40S! rAsubunit! shortage!(Figure( 3).!Analysis!of!preArRNA!processing!by!northern!blotting!and!pulseA chase!labelling!analysis!clearly!indicates!that!the!40S!rAsubunit!deficit!exhibited!by!the! rps12!mutant!is!due!to!a!strong!inhibition!of!processing!of!the!20S!preArRNA,!which! leads!to!reduced!formation!of!mature!18S!rRNA.!The!delay!in!20S!preArRNA!processing! of!rps12∆!cells!results!from!reduced!cleavage!of!cytoplasmic!20S!preArRNA!at!site!D! since!20S!preArRNA!was!accumulated!in!the!cytoplasm!and!export!of!preA40S!particles! was!not!impaired,!as!we!did!not!observe!nuclear!accumulation!of!the!SSU!reporters!in! rps12∆!cells!(Figures(6(and(7).!In!a!similar!way,!it!has!been!described!that!human!eS12! is!required!in!the!conversion!of!18SAE!to!18S,!homologous!to!the!20S!preArRNA!to!18S! rRNA!yeast!processing!(O'Donohue!et!al.,!2010).!Moreover,!preArRNA!processing!at!site! A2!seems!to!be!slightly!delayed!as!suggested!by!the!decrease!of!27S!preArRNA!and!the! appearance!of!the!aberrant!21S!preArRNA!upon!depletion!of!yeast!eS12!(Figures(4(and( 5).!! The!effects!of!the!RPS12#deletion!are!very!similar!to!those!shown!and!previously! ! ! 102( Chapter 2 reported!for!the!depletion!of!its!neighbour!rAprotein!eS31.!The!rAprotein!eS31!is!also!a! quasiAessential!protein!located!on! the! beak! of! the! small! rAsubunit.!Absence!of!eS31! causes!a!strong!40S!rAsubunit!shortage!due!to!delayed!preArRNA!cleavage!at!sites!A2! and!cytoplasmic!maturation!of!20S!preArRNA!at!site!D,!similarly!to!the!absence!of!eS12! (Figures( 3( and( 5)((Finley! et! al.,! 1989;! Lacombe! et! al.,! 2009).! We! have! shown! that! simultaneous!deletion!of!both!genes!did!not!enhance!these!phenotypes.! Similar!defects!in!preArRNA!processing!have!been!reported!for!different!rAprotein! mutants,! especially! for! those! rAproteins! located! on! the! head! of! the! small! rAsubunit! (FerreiraACerca!et!al.,!2005;!FerreiraACerca!et!al.,!2007).!However,!it!is!not!clear!how! might!rAproteins!located!on!the!beak!affect!the!conversion!of!20S!preArRNA!to!mature! 18S! rRNA.! Due! its! positions,! it! seems! unlikely! that! eS12! or! eS31! directly! catalyses! cleavage! of! 20S! preArRNA! in! yeast! ribosomes.! Two! explanations! are! possible.! First,! these!rAproteins!could!be!important!to!establish!the!conformation!of!the!3’︎!end!of!20S! preArRNA!required!for!efficient!cleavage!at!site!D!indirectly!through!general!effects!on! 40S!rAsubunit!structure.!Analysis!of!the!structure!of!small!ribosomal!subunits!suggests! a!general!effect!in!subunit!structure!since!eS12!neither!eS31!are!not!close!enough!to! interact!with!sequences!in!ITS1.!Second,!it!is!also!possible!that!eS12!and!eS31!were! necessary!to!recruit!or!position!nucleases!or!other!cofactors,!such!as!Nob1,!required! for! cleavage! of! 20S! preArRNA.! Further! experiments! are! required! to! check! if! such! cofactors!are!present!in!preAribosomal!particles!in!absence!of!eS12!or!eS31!to!address! the!precise!effect!both!rAproteins!in!preArRNA!processing.! On!the!other!hand,!preA40S!rAparticles!containing!the!20S!preArRNA!present!in! rps12∆!mutant!cells!are!not!competent!for!translation!elongation!since!the!20S!preA rRNA!accumulated!in!these!cells!was!only!found!to!associate!with!40S!rAsubunits!and! 80S! ribosomes,! but! not! with! translating! ribosomes! (Figure( 8).! However,! we! cannot! exclude!the!possibility!that!subunits!containing!the!20S!preArRNA!are!at!least!partially! active! in! protein! synthesis! because! small! amounts! of! this! precursor! coAsedimented! with!polysomes.!By!contrast,!20S!preArRNAAcontaining!40S!rAsubunits!could!engage!in! translation! upon! depletion! of! eS31.! Although! preA40S! rAparticles! are! not! found! in! polysomes!in!wildAtype!cells!or!in!most!mutants!that!accumulate!cytoplasmic!20S!preA rRNA!(e.g.(Ford!et!al.,!1999;!Jakovljevic!et!al.,!2004;!Strunk!et!al.,!2012)),!presence!of! ! 103( Chapter 2 20S!preArRNA!in!ribosomeApolysome!fractions!is!also! observed!when!the!NAterminal! ubiquitin!moiety!of!eS31!was!deleted,!or!when!some!rAproteins!or!ribosome!biogenesis! factors!were!depleted!(e.g.!(GarcíaAGómez!et!al.,!2014;!Lacombe!et!al.,!2009)).!! Probably,! qualitative! differences! between! 40S! rAsubunits! containing! 20S! preA rRNA!in!rAparticles!lacking!eS12!or!eS31!could!be!found.!Moreover,!it!seems!that!these! 40S! rAsubunits! containing! the! 20S!preArRNA! present! in! rps12∆! and! ubi3∆!cells! were! altered!in!such!a!way!that!they!affect!the!correct!function!of!the!translation!machinery! since!both!strains!are!defective!in!translation!elongation!(Figure(12).(In!this!sense,!we! had!previously!reported!that!the!truncation!of!the!NAterminal!extension!of!eS31,!which! reaches!the!AAsite!of!the!40S!rAsubunit,!affects!the!fidelity!of!translation!(FernándezA Pevida!et!al.,!2016).! Finally,!we!have!also!analysed!the!connection!between!eS12,!eS31!and!ribosomal! biogenesis!factors!Enp1!and!Ltv1!to!further!unravel!how!eS12!and!eS31!contribute!to! the!biogenesis!of!40S!rAsubunits.!These!factors!are!assembled!close!eS12!and!eS31!and! both!are!important!for!beak!formation!(Scaiola!et!al.,!2018).!The!transAacting!factor! Enp1!is!already!present!in!90S!preAribosomes,!whereas!Ltv1!binds!directly!to!Enp1!later! in! preA40S! rAparticles! (Schäfer! et! al.,! 2003).! In! the! cytoplasm,! both! factors! forms! a! complex!that!is!phosphorylationAdependent!released!to!permit!the!final!remodelling!of! the!head!region!of!40S!rAsubunits!(Collins!et!al.,!2018).!Moreover,!mutations!related! with!both!factors!lead!to!defects!in!ribosome!biogenesis!that!resembled!those!seen!in! strains!genetically!depleted!of!rAproteins!eS12!and!eS31!(Chen!et!al.,!2003;!Seiser!et!al.,! 2006).!Thus,!transAacting!factors!Enp1!and!Ltv1!could!be!somehow!link!with!eS12!or! eS31.! In! agreement! with! this! hypothesis,! we! have! found! genetic! interaction! between!the!absence!of!either!eS12!or!eS31!and!Ltv1!or!Enp1!(Figure(9).!However,!this! interaction!it!seems!not!to!be!related!with!the!incorporation!or!release!of!these!factors! into!preA40S!rAparticles,!since!we!had!not!found!significant!changes!in!the!subcellular! localization!of!these!transAacting!factors!upon!eS12!or!eS31!depletion!(Figure(10).!On! the!other!hand,!rAprotein!eS12!apparently!is!not!required!to!eS31!assembly!as!suggest! the! cytoplasmic! signal! of! eS31AGFP! in! rps12∆!cells! (Figure( 6).!Clearly,! future! experiments!are!required!in!order!to!understand!the!relation!between!eS12!and!eS31! and!transAacting!factors.!! ! ! 104( Chapter 2 In! conclusion,! our! data! clearly! indicate! that! formation! of! the! beak! is! a! preA requisite! for! the! efficient! maturation! of! 40S! rAsubunits! and! its! proper! function.! However,! some! aspects! as! the! timing! of! eS12! assembly! remain! unexplored.! Some! analyses!suggest!that!rAproteins!that!form!the!head!domain!of!the!40S!rAsubunits!are! the!latest!rAproteins!assembled!during!ribosome!biogenesis!(e.g.!(FerreiraACerca!et!al.,! 2007;! O'Donohue! et! al.,! 2010)).! In! this! sense,! rAproteins! located! in! the! head! are! important!for!downstream!maturation!steps!as!the!nucleoAcytoplasmic!export!of!preA 40S!rAparticles!and!processing!of!late!preArRNAs!(de!la!Cruz!et!al.,!2015;!FerreiraACerca! et!al.,!2007;!O'Donohue!et!al.,!2010).!In!agreement,!a!predominantly!late!nucle(ol)ar! assembly! of! eS31! has! been! suggested! (FernándezAPevida! et! al.,! 2016).! Taking! into! account! the! defects! in! preArRNA! processing! in! rps12∆!cells,! we! could! assumed! that! stable!assembly!of!eS12!occurs!at!a!relatively!late!stage!during!ribosome!biogenesis.! Surprisingly,!in!recent!cryoAEM!studies!the!beak!structure!of!the!head!region!seems!to! be!practically!molded!in!the!early!90S!preAribosomal!particles!(Sun!et!al.,!2017).!In!that! sense,! we! cannot! rule! out! the! possibility! of! an! early! association! in! preAribosomal! particles!of!rAproteins!eS12!and!eS31!in!an!immature!conformation!considering!that! the!head!domain!region!is!reorganized!before!20S!preArRNA!cleavage,!40S!rAproteins! interact! particularly! weakly! with! 90S! preAribosomal! particles,! and! a! conformational! change! of! the! NAterminus! of! eS31! has! been! described! in! human! preA40S! rAparticles! (Ameismeier!et!al.,!2018;!Schäfer!et!al.,!2006).!!Thus,!further!experiments!are!required! to!address!the!precise!timing!of!eS12!assembly!into!preAribosomal!particles.! ! ( ( ! 105( Chapter 2 MATERIALS(AND(METHODS( ( Strains(and(microbiological(methods( All! yeast! strains! used! in! this! work! are! listed! in! Table( 1.! Unless! otherwise! indicated,! experiments! were! conducted! in! the! W303! genetic! background.! Strains! SMY227!and!SMY231!are!meiotic!rps12∆!and!ubi3∆!null!segregants!of!strains!Y21666! and!Y24116!(Euroscarf),!respectively.!Strain!SMY315!was!generated!by!PCRAbased!gene! disruption!of!the!RPS12!gene!in!the!diploid!W303!strain.!To!this!end,!we!used!genomic! DNA! of! the! SMY227! strain,! which! harbours! a! genomic! rps12::kanMX4! allele,! as! a! template!and!the!oligonucleotide!pair!EcoRI_S12up!and!S12_check_down!(see!Table( 3).! Transformants! were! selected! on! YPD! plates! containing! 200! µg/ml! G418,! some! candidates! were! chosen! and! the! correctness! of! the! integration! verified! by! PCR.! Positive!heterozygous!candidates!were!transformed!with!the!YCplac33ARPS12!plasmid,! then!sporulated,!and!tetrads!were!dissected.!SMY315!was!selected!as!a!representative! rps12∆!null!haploid!mutant.! ! To! generate! SMY372,! TLY14.3C! was! crossed! to! SMY315! [YCplac33ARPS12];! the! resulting! diploid! was! sporulated,! and! tetrads! were! dissected.! Spore! clones! from! different!tetratypes!were!obtained!and!restreaked!on!5AFOAAcontaining!plates.!Other! strains,!listed!in!Table(1,!harbouring!combinations!of!different!alleles!were!obtained!by! a!similar!approach.!! ! Growth!and!handling!of!yeast!and!yeast!media!were!done!following!established! procedures! (Kaiser! et! al.,! 1994).! Strains! were! grown! at! the! indicated! temperatures! either!in!rich!YPD!medium!(1%!yeast!extract,!2%!peptone!and!2%!glucose)!or!synthetic! minimal! medium! (0.15%! yeast! nitrogen! base,! 0.5%! ammonium! sulphate)! supplemented! with! the! appropriate! amino! acids! and! bases! as! nutritional! requirements,!and!containing!2%!glucose!(SD)!as!carbon!source.!To!prepare!plates,!2%! agar!was!added!to!the!media!before!sterilization.!Yeast!cells!were!transformed!by!a! lithium!acetate!method!(Gietz!et!al.,!1992).!Tetrads!were!dissected!with!a!MSM200! micromanipulator!(Singer!Instruments,!UK).!Escherichia#coli!strain!DH5α!was!used!for! ! ! 106( Chapter 2 cloning! and! propagation! of! plasmids.! All! recombinant! DNA! techniques! were! done! according!to!established!procedures!(Sambrook!et!al.,!1989).! ! Plasmids( Plasmids!used!in!this!work!are!listed!in!Table(2.!To!generate!YCplac33ARPS12,!a! PCR!was!performing!using!yeast!genomic!DNA!as!template!and!the!oligonucleotides! BamHI_S12up! and! S12_SacIdown! (Table( 3),! placed! plusAminus! 1! kb! upstream! and! downstream!from!the!startAstop!codon!of!the!RPS12!ORF.!The!ca.!2.4!kb!PCR!product! was!restricted!with!BamHI!and!SacI!and!cloned!into!YCplac33,!which!was!also!digested! with!the!same!enzymes.!The!rest!of!plasmids!have!been!reported!elsewhere!(see!Table( 2).! ! Sucrose(gradient(centrifugation( Polysome! and! ribosomal! subunit! analyses! were! performed! as! previously! described!(Kressler!et!al.,!1997).!Ten!A260!of!total!cell!extract!were!load!onto!7A50%! sucrose! gradients.! These! gradients! were! centrifuged! at! 39,000! rpm! in! a! Beckman! Coulter!rotor!SW41!for!2!h!45!min!(for!polysome!profile!analyses)!or!for!4!h!30!min!(for! ribosomal!subunit!analyses);!then,!the! A254!was!continuously!monitor!using!an!ISCO! UAA6!system.! ! When!required,!fractions!of!0.5!ml!were!collected!from!the!gradient.!RNA!was! extracted!from!each!fraction!and!an!equal!volume!of!RNA!from!individual!fractions!was! subjected!to!northern!analyses!(see!below)!as!previously!described!(de!la!Cruz!et!al.,! 1998a).! ! RNA(extractions(and(northern(hybridization(analyses( Total! RNA! was! extracted! from! samples! corresponding! to! 10! OD600! units! of! exponentially! grown! cells! by! the! acidAphenol! method! (Ausubel! et! al.,! 1994).! Equal! amounts! of! total! RNA! were! loaded! on! 1.2%! agaroseA6%! formaldehyde! or! on! 7%! polyacrylamideA8M!urea!gels!as!previously!described!(Venema!et! al.,! 1998).!Specific! antisenseAoligonucleotides! (Table( 3)! were! used! to! detect! specific! preA! and! mature! rRNAs.!Oligonucleotides!were!5’Aend!labelled!with![γA32P]ATP! (6000! Ci/mmol;!Perkin! ! 107( Chapter 2 Elmer)! as! previously! described! (Venema! et! al.,! 1998).! Phosphorimager! analysis! was! performed! with! a! Typhoon™! FLAA9000! imaging! system! (GE! Healthcare)! and! signals! intensities! were! quantified! using! the! GelQuant.NET! software! (biochemlabsolutions.com).! ( Pulse]chase(labelling(of(pre]RNA( For! [5,6A3H]uracil! pulseAchase! labelling! analysis,! strains! W303A1A! and! SMY315! were!first!transformed!with!an!empty!YCplac33!plasmid!to!make!them!prototrophic!for! uracil.!Transformants!were!grown!in!50!ml!of!liquid!SDAUra!medium!to!midAlog!phase! at! 30! ºC.! Then,! the! cells! were! concentrated! in! 1! ml! of! SDAUra! medium! and! pulseA labelled!using!100!µCi!of![5,6A3H]uracil!(45!to!50!Ci/mmol;!Perkin!Elmer)!per!40!OD600! units!of!yeast!cells!as!exactly!described!in!Kressler!et#al.!(Kressler!et!al.,!1998).!Cells! were!chased!for!5,!15,!30!and!60!min!after!diluting!200!µl!aliquots!of!pulseAlabelled! cells!in!4!ml!of!SD!medium!containing!an!excess!(1!mg/ml)!of!nonAradioactive!uracil.! Total!RNA!was!extracted!by!the!acidAphenol!method!as!above.!!! ! Uracil!incorporation!was!measured!by!scintillation!counting!and!3000!cpm!per! RNA! sample! was! loaded! and! resolved! on! 1.2%! agarose–6%! formaldehyde! and! 7%! polyacrylamideA8M! urea! gels.! RNA! was! then! transferred! to! HybondAN! nylon! membranes!(GE!Healthcare),!crosslinked!to!the!membranes,!and!exposed!to!a!tritium! screen! (BASAIP! TR2040E;! GE! Healthcare).! Visualization! was! performed! with! a! Typhoon™!FLAA9000!imaging!system!(GE!Healthcare).! ! Detection(of(fluorescent(proteins(by(microscopy( To!test!preAribosomal!particle!export,!the!appropriate!strains!were!transformed! with! pRS315ARPL25AeGFPANOP1AmRFP,! pRS315ARPS3AeGFPANOP1AmRFP! (gifts! from! J.! Bassler!and!E.!Hurt),!YCplac111ARPS10AyEGFP!or!YCplac111AUBI3AyEGFP!plasmids!(see! Table(2).!Transformants!were!grown!in!selective!SD!medium.!Cells!were!washed!and! resuspended!in!PBS!buffer!(140!mM!NaCl,!8!mM!Na2HPO4,!1.5!mM!KH2PO4,!2.75!mM! KCl,!pH!7.3)!before!their!microscopy!inspection.!When!required,!Hoechst!was!used!to! stain! nuclear! DNA.! Cells! were! examined! with! an! Olympus! BX61! fluorescence! ! ! 108( Chapter 2 microscope!equipped!with!a!digital!camera;!images!were!analysed!using!the!CellSens! software!(Olympus).! ! To! study! the! subcellular! location! of! GFPAtagged! Enp1,! Ltv1! and! Ltv1∆NES,! the! appropriate!strains!(see!Table( 1)!were!grown!in!YPD!medium.!Hoechst!was!used!to! stain! DNA! in! the! nucleus.! Fluorescently! labelled! cells! were! inspected! as! described! above.! ! Fluorescence(in+situ(hybridization(microscopy( To! examine! the! localization! of! the! 20S! preArRNA,! fluorescence! in# situ! hybridisation! (FISH)! was! performed! in! spheroblasted! cells! fixed! with! formaldehyde,! as! previously! described!(Grosshans!et!al.,!2000;!RodríguezAGalán!et!al.,!2015).!A!Cy3Alabelled!ITS1A specific! probe! (see! Table( 3)! was! used.! Cells! were! also! stained! with! DAPI! (4,6A diamidinoA2Aphenylindole)! to! visualize! DNA.! Cells! were! examined! by! fluorescence! microscopy!as!described!above.! ! Quantification(of(translation(accuracy.( To! measure! the! efficiency! of! translation! termination! and! of! amino! acid! misincorporation,!the!strains!W303A1A,!SMY315!and!TLY14.3C!were!transformed!with! the!different!dualAluciferase!reporter!plasmids!generously!provided!by!D.!Bedwell!(see! Table(2).!Luciferase!activities!were!measured!as!previously!described!(SalasAMarco!and! Bedwell,! 2005)!using! the! DualAGlo®! Luciferase! Assay! System! (Promega).! Cells! from! each!strain!were!grown!in!liquid!SDAUra!medium!to!midAlog!phase!at!30ºC!and!firefly! and!Renilla!luciferase!luminescence!levels!were!measured!at!room!temperature!with!a! CLARIOstar! 1.20! microplate! reader! (BMG! Labtech,! Germany)! adjusts! to! readtype! endpoint!and!default!settings.!Assays!were!done!in!quadruplicate!and!the!data!were! expressed! as! the! mean! ±! the! standard! deviation.! Error! rates! for! each! strain! were! calculated!as!the!percentage!of!the!firefly/Renilla!luciferase!activity!(mutant!plasmid)! divided!by!the!firefly/Renilla!luciferase!activity!(wildAtype!plasmid).! ! ! ! 109( Chapter 2 Table(1.(Yeast(strains( ! ( Strain( ( Relevant(genotype( ( Source( ! BY4741! ! MATa!his3∆1#leu2∆0#met15∆0#ura3∆0! ! Euroscarf! Y21666! MATa/MATα!his3∆1/his3∆1!leu2∆0/leu2∆0!met15∆0/MET15! LYS2/lys2∆0#ura3∆0/ura3∆0#RPS12/rps12::kanMX4! Euroscarf! Y24116! MATa/MATα!his3∆1/his3∆1!leu2∆0/leu2∆0!met15∆0/MET15! LYS2/lys2∆0#ura3∆0/ura3∆0#UBI3/ubi3::kanMX4# Euroscarf! SMY227! As!BY4741!but!rps12::kanMX4! This!work! SMY231! As!BY4741!but!ubi3::kanMX4! This!work! W303A1A! MATa!ade221#his3211,15#leu223,112#trp121#ura321! (Thomas!and! Rothstein,! 1989)! W303A1B! As!W303A1A!but!MATα! (Thomas!and! Rothstein,! 1989)! SMY315! As!W303A1B!but!rps12::kanMX4! This!work! TLY14.3C! As!W303A1A!but!ubi3::HIS3MX6! (Lacombe!et!al.,! 2009)! SMY372! As!W303A1B!but!rps12::kanMX4!ubi3::HIS3MX6! This!work! YGM96! As!W303A1A!but!ENP1AGFP::HIS3MX6! (Moriggi!et!al.,! 2015)! YGM131! As!W303A1A!but!LTV1AGFP::natNT2!ade3::kanMX4! D.!Kressler's!lab! YGM138! As!W303A1A!but!LTV1∆NESAGFP::natNT2!ade3::kanMX4! D.!Kressler's!lab! SMY443! As!W303A1A!but!rps12::kanMX4!ENP1AGFP::HIS3MX6! This!work! SMY447! As!W303A1A!but!rps12::kanMX4!LTV1AGFP::natNT2! This!work! SMY452! As!W303A1A!but!rps12::kanMX4!LTV1∆NESAGFP::natNT2! This!work! SMY456! As!W303A1A!but!ubi3::HIS3MX6!ENP1AGFP::HIS3MX6! This!work! SMY459! As!W303A1A!but!ubi3::HIS3MX6!LTV1AGFP::natNT2! This!work! SMY466! As!W303A1A!but!ubi3::HIS3MX6!LTV1∆NESAGFP::natNT2! This!work! SMY307! As!W303A1A!but!ltv1::hphNT1!! B.!Pertschy's!lab! SMY353! As! W303A1A! but! rps12::kanMX4! ltv1::HIS3MX6! [YCplac33A RPS12]! This!work! SMY345! As! W303A1A! but! ubi3::HIS3MX6! ltv1::hphNT1! [YCplac33A UBI3]! This!work! SMY310! As!BY4742!but!enp1::kanMX4![pRS316AENP1]! E.!Hurt's!lab! SMY431! As!BY4741!but!rps12::kanMX4!enp1::kanMX4![pRS316AENP1]! This!work! SMY439! As!BY4741!but!ubi3::HIS3MX6!enp1::kanMX4![pRS316AENP1]! This!work! ! ! ! ! ! 110( Chapter 2 Table(2.(Plasmids(used(in(this(work( ! Name((collection(name)( Relevant(information( Reference( ! YCplac33! ! CEN,!URA3!! (Gietz!and! Sugino,!1988)! YCplac111! CEN,!LEU2!! (Gietz!and! Sugino,!1988)! YCplac33ARPS12! eS12;!CEN,!URA3!! This!work! YCplac33AUBI3! Ubi3;!CEN,!URA3!! (Lacombe!et! al.,!2009)! pRS315ARPL25AeGFPA NOP1AmRFP! uL23AeGFP,!Nop1AmRFP;!CEN,!LEU2! (Ulbrich!et!al.,! 2009)! pRS315A!RPS3AeGFPA NOP1AmRFP! uS3AeGFP,!Nop1AmRFP;!CEN,!LEU2!! (Ulbrich!et!al.,! 2009)! YCplac111AUBI3AyEGFP! (pDK4516)! Ubi3AyEGFP;!CEN,!LEU2! (FernándezA Pevida!et!al.,! 2016)! YCplac111ARPS10AA yEGFP! RPS10AAyEGFP;!CEN,!LEU2! (FernándezA Pevida!et!al.,! 2016)! YEplac195AlucCAAA! (pDB688)! Renilla!and!Firefly!construct!containing!a!CAC! codon!control!(H245),!2µ,!URA3.!! (SalasAMarco! and!Bedwell,! 2005)! YEplac195AlucCAAA!H245! CGC!(pDB868)! Renilla!and!Firefly!construct!containing!a!CGC! codon!(H245R),!2µ,!URA3.!! (SalasAMarco! and!Bedwell,! 2005)! YEplac195AlucCAAC! (pDB722)! Renilla!and!Firefly!construct!containing!a!CAA! codon!control,!2µ,!URA3.! (Keeling!et!al.,! 2004)! YEplac195AlucUAAC! (pDB723)! Renilla!and!Firefly!construct!containing!a!UAA! stop!codon,!2µ,!URA3.!! (Keeling!et!al.,! 2004)! YEplac195AlucCGAC! (pDB690)! Renilla!and!Firefly!construct!containing!a!CGA! codon!control,!2µ,!URA3.!! (Keeling!et!al.,! 2004)! YEplac195AlucUGAC! (pDB691)! Renilla!and!Firefly!construct!containing!a!UGA! stop!codon,!2µ,!URA3.! (Keeling!et!al.,! 2004)! YEplac195AlucCAGC! (pDB721)! Renilla!and!Firefly!construct!containing!a!CAG! codon!control,!2µ,!URA3.! (Keeling!et!al.,! 2004)! YEplac195AlucUAGC! (pDB720)! Renilla!and!Firefly!construct!containing!a!UAG! stop!codon,!2µ,!URA3.# (Keeling!et!al.,! 2004)! YCplac111ALTV1! Ltv1;!CEN,!LEU2# (Mitterer!et! al.,!2016)! YCplac111Altv1A S336/S339>A! Ltv1[S336A!S339A];!CEN,!LEU2!! (Mitterer!et! al.,!2016)! YCplac111Altv1A S336/S339/S342>A! Ltv1[S336A!S339A!S342A];!CEN,!LEU2!! (Mitterer!et! al.,!2016)! YCplac111Altv1AS6>A! Ltv1[S336A!S339A!S342A!S344A!S345A!S346A];! CEN,!LEU2! (Mitterer!et! al.,!2016)! pRS316AENP1! Enp1;!CEN,!URA3! (Chen!et!al.,! 2003)! pRS315! CEN,!LEU2! (Sikorski!and! Hieter,!1989)! ! 117( Discussion general!ribosomeAassociated!chaperones!NAC/SsbARAC!may!not!be!enough!to!ensure! the! proper! folding! of! these! rAproteins.! Thus,! an! additional! strategy! such! as! fusing! ubiquitin! to! eS31! and! eL40! could! help! these! rAproteins! to! acquire! its! correct! conformation.! ! In!conclusion,!the!use!of!amenable!tools!on!yeast!biochemistry!and!genetics!is! allowing!us!to!unveil!the!biological!role!of!ubiquitin!fused!to!rAproteins!eL40!and!eS31.! Our!findings!provide!evidence!of!how!ubiquitin!could!contribute!to!the!expression!of!rA proteins!eL40!and!eS31,!their!further!assembly!into!preAribosomal!particles!and!their! function.!With!the!exception!of!the!protozoan!parasite!Giardia#lamblia,!there!is!so!far! no!evidence!of!a!naturally!occurring!nonAcleaved!form!of!ubiquitin!fused!to!eS31!or! eL40!as!constituent!of!mature!ribosomes!(Catic!et!al.,!2007),!thus!proteolytic!cleavage! of!the!ubiquitin!moiety!of!the!precursors!seems!to!be!a!general!requirement!for!the! ribosomal!functionality!of!these!two!rAproteins.!Instead,!ubiquitin!may!act!as!a!in#vivo! molecular!chaperone!for!efficient!synthesis!of!eL40!and!eS31,!a!function!that,!although! partially,!could!artificially!fulfil!the!yeast!ubiquitinAlike!Smt3!protein.!In!support!to!this! cellular!function,!ubiquitin!fused!to!eS31!has!evolutionarily!diverged!into!ubiquitinAlike! proteins!in!some!organisms!such!as!Caenorhabditis#elegans!or!C.#briggsae!(Jones!and! Candido,!1993).!However,!some!aspects!remain!opened!yet.!Why!are!eL40!and!eS31! the!sole!rAproteins!fused!to!ubiquitin?!It!could!serve!to!maintain!the!stoichiometry!of! 60S! and! 40S! rAsubunits?! Once! the! ubiquitin! is! cleaved,! it! remains! nonAcovalently! attached!to!eL40!and!eS31?!Could!be!Ubi1/2!the!source!of!ubiquitin!for!the!cytoplasm! and! Ubi3! for! the! nucleus?! Future! experiments! are! required! in! order! to! understand! exactly!when!and!how!the!cleavage!of!ubiquitin!precursors!takes!place!and!hence,!to! better!clarify!how!rAproteins!eL40!and!eS31!contribute!to!ribosome!biogenesis.! ! In! this! sense,! although! some! functional! aspects! of! rAprotein! eS31! has! been! previously!studied!(FerreiraACerca!et!al.,!2005;!FerreiraACerca!et!al.,!2007;!Finley!et!al.,! 1987;!Lacombe!et!al.,!2009),!in!Chapter(2!we!have!analysed!in!more!details!the!role!of! this!protein!in!ribosome!biogenesis!and!function,!in!the!context!of!the!beak!region!of! 40S! rAsubunit.! To! this! end,! we! have! generated! and! analysed! mutants! lacking! the! rA proteins! eS12! and! eS31,! alone! or! in! combination.! Both! proteins! are! located! on! the! ! ! 118( Discussion beak!of!the!small!rAsubunit,!a!eukaryoticAspecific!structure!that!has!been!transformed! from!the!helix!H33!found!in!prokaryotic!ribosomes.!Our!analysis!is!the!first!report!in! which!eS12!has!been!characterized!and!hence,!the!first!study!about!the!contribution!of! the!rAproteins!of!the!beak!to!ribosome!biogenesis!and!function.! ! Herewith,!we!have!analysed!the!consequences!for!ribosome!biogenesis!of!the! deletion!of!RPS12!and!UBI3#genes,!which!codes!for!the!quasiAessential!proteins!eS12! and! eS31,! respectively.! Interestingly,! deletion! of! any! of! these! genes! results! in! very! similar!severe!defects!in!ribosome!biogenesis:!a!strong!deficit!in!40S!rAsubunit!and!an! impairment!of! cytoplasmic! 20S!preArRNA! processing! at!site! D.! Moreover,!preA40S! rA particles!containing!the!20S!preArRNA!in!rps12∆!and!ubi3∆!cells!can!assemble!into!80S! ribosomes!but!inefficiently!engage!in!translation.!On!the!other!hand,!ribosomes!lacking! eS12!or!eS31!are!slightly!defective!in!translation!accuracy.!The!important!role!of!eS12! and! eS31! in! ribosome! biogenesis! is! also! inferred! by! the! evidence! that! a! genetic! interaction!between!the!absence!of!either!eS12!or!eS31!and!that!the!ribosomal!transA acting!factors!Ltv1!or!Enp1!has!been!found.!Furthermore,!deletion!of!RPS12!or!UBI3# leads!to!similar!levels!of!rAprotein!aggregation!(see(Figure(9,(chapter(1).! ! Considering!the!above!results,!we!conclude!that!rAproteins!eS12!and!eS31!display! similar!roles!in!ribosome!biogenesis!and!hence,!the!beak!domain!is!important!for!40S! rAsubunit!maturation!and!function.!This!work!opens!new!research!lines!to!study!some! aspects! that! remain! undiscovered! yet! regarding! the! role! of! these! proteins.! For! example,! further! experiments! are! required! to! understand! when! and! how! eS12! is! assembled!into!preAribosomal!particles,!to!determine!whether!the!assembly!of!eS12! depends!on!some!rAproteins!like!eS31!and!to!better!define!the!connection!between! eS12!and!eS31!and!the!transAacting!factors!Ltv1!and!Enp1.! ! In!summary,!the!studies!carried!out!in!this!Thesis!Work!represents!a!modest!but! relevant!contribution!to!our!knowledge!about!the!function!of!rAproteins!in!ribosome! assembly.!As! we!mentioned!above,!ribosomopathies!are!linked!to!ribosome! defects! caused! by! mutations! in! rAprotein! genes,! so! understanding! the! exact! functions! of! rA proteins!during!ribosome!assembly!is!a!very!pertinent!issue.!Moreover,!one!of!these! ! 119( Discussion pathologies,! the! DiamondABlackfan! anemia,! has! been! linked! with! mutations! in! rA proteins! especially! of! the! small! rAsubunit! (Narla! and! Ebert,! 2010).! Interestingly,! mutations!in!human!eS31!and!eS10,!neighbours!to!eS12,!seem!to!be!related!with!the! DiamondABlackfan! anemia! (Doherty! et! al.,! 2010;! Gazda! et! al.,! 2008).! ! Thus,! our! progress!in!understanding!the!role!of!eS12!and!eS31!in!ribosome!biogenesis!can!help! us!to!clarify!the!underlying!physiopathological!mechanisms!of!these!diseases.! ! 6.(CONCLUSIONS( ! ! ! ! ! ! ! ! ! 122( Conclusions ! ! 1. The!NAterminal!ubiquitin!moiety!fused!to!rAprotein!eS31!and!eL40!is!required! for!the!efficient!production!of!eS31!and!eL40,!respectively.! 2. The!ubiquitin!moiety!of!Ubi1! is!not!strictly!needed!for!the!assembly! of!eL40! into!preA60S!rAparticles.!! 3. The!ubiquitinAlike!protein!SUMO!could!artificially!replace!the!ubiquitin!moiety! of!Ubi1,!which!increases!the!expression!and!solubility!of!eL40A.! 4. Ribosomal! proteins! of! the! beak! eS12! and! eS31! are! quasiAessential! proteins! required!for!cell!growth!and!synthesis!of!40S!rAsubunits.! 5. Ribosomal!proteins!eS12!and!eS31!are!necessary!for!efficient!cytoplasmic!20S! preArRNA!processing!but!are!dispensable!for!preA40S!rAparticles!export!from!the! nucleus.! 6. Both!ribosomal!proteins!of!the!beak!are!required!for!translation!accuracy.! ! ! ! ! 6.(BIBLIOGRAPHY( ! ! ! ! ! 126( Bibliography Albanèse,! V.,! Yam,! A.Y.,! Baughman,! J.,! Parnot,! C.,! and! Frydman,! J.! (2006).! Systems! analyses!reveal!two!chaperone!networks!with!distinct!functions!in!eukaryotic!cells.!Cell# 124,!75A88.! Alksne,!L.E.,!Anthony,!R.A.,!Liebman,!S.W.,!and!Warner,!J.R.!(1993).!An!accuracy!center! in!the!ribosome!conserved!over!2!billion!years.!Proc.!Natl.!Acad.!Sci.!USA#90,!9538A 9541.! Ameismeier,! M.,! Cheng,! J.,! Berninghausen,! O.,! and! Beckmann,! R.! (2018).! Visualizing! late!states!of!human!40S!ribosomal!subunit!maturation.!Nature#558,!249A253.! Amerik,! A.Y.,! and! Hochstrasser,! M.! (2004).! Mechanism! and! function! of! deubiquitinating!enzymes.!Biochim.!Biophys.!Acta#1695,!189A207.! 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