Estudio del ensamblaje ribosómico en Saccharomyces Cerevisae.

Estudio del ensamblaje ribosómico en Saccharomyces cerevisiae Francisco José Espinar Marchena Tesis doctoral 2017 Departamento de Genética Universidad de Sevilla Estudio del ensamblaje ribosómico en Saccharomyces cerevisiae Memoria presentada para aspirar al grado de Doctor en Biología Vo Bo: El director de la Tesis El doctorando Jesús de la Cruz Díaz Francisco José Espinar Marchena ÍNDICE INTRODUCCIÓN……………………………………………………………………………… 1  El Ribosoma. Estructura, función y evolución…………………………………….. 3  Síntesis de ribosomas en S. cerevisiae……………………………………………. 7  Procesamiento de los rRNAs……………………………………………………….. 8  Las proteínas ribosómicas en S. cerevisiae……………………………………... 12  Maduración de la partícula pre-ribosómica temprana 90S…………………….. 16  El ensamblaje de la subunidad 40S………………………………………………. 20  El ensamblaje de la subunidad 60S………………………………………………. 24  Mecanismos de vigilancia en la biogénesis del ribosoma…………………….. 31 OBJETIVOS………………………...……………………………………………………….. 35 CAPÍTULO 0 – Las proteínas ribosómicas L14 y L16 del ribosoma de Saccharomyces cerevisiae………….…………………………………………………… 39 CAPÍTULO 1 – Role of the yeast ribosomal protein L16 in ribosome biogenesis……………………………………………………………………… 45  INTRODUCTION……………………………………………………………………. 47  RESULTS……………………………………………………………………………. 49  Depletion of L16 leads to a strong 60S r-subunit shortage……………… 49  Depletion of L16 impairs pre-rRNA processing and induces the turnover of 27S pre-rRNAs………………………………………………….. 51  Depletion of L16 impairs export of pre-60S r-particles from the nucleus to the cytoplasm……………………………………………………. 53  L16 assembles within early pre-ribosomal particles……………………… 54  The eukaryote-specific C-terminal extension of L16 is required for growth……………………………………………………………………… 56  Importance of the C-terminal extension of L16 in 60S r-subunit biogenesis…………………………………………………………. 58  EXPERIMENTAL PROCEDURES………………………………………………... 63 CAPÍTULO 2 – Ribosomal protein L14 contributes to the early assembly of 60S ribosomal subunit in Saccharomy cescerevisiae……….............................… 67  INTRODUCTION……………………………………………………………………. 69  RESULTS……………………………………………………………………….…… 75  Yeast L14 assembles in the nucle(ol)us within early pre-60S r-particles……………………………………………………………………... 75  L14 is required for normal accumulation of 60S ribosomal subunits…… 78  L14 is required for 27S pre-rRNA processing and normal production of 25S and 5.8S rRNAs………………………………………… 81  Depletion of L14 affects negatively 27S pre-rRNA processing……...….. 82  Depletion of L14 leads to nuclear retention of pre-60S ribosomal particles………………………………………………………….... 84  L14 is required for the stable assembly of 60S r-proteins located at its immediate neighbouring r-proteins and surrounding the polypeptide exit tunnel………………………………………………………. 85  L14 is required for the stable association of a set of 60S r-subunit biogénesis factors required for processing of 27SB pre-rRNA…………………………………………………………………….... 88  Disruption of the interaction between the eukaryote-specific C-terminal extensions of L14 and L16 r-proteins causes paromomycin hypersensitivity………………………………………………. 89  EXPERIMENTAL PROCEDURES.………………………………………….……. 91 DISCUSIÓN……………………………………………………………………………..…… 99 CONCLUSIÓNES……………………………………………………………..…………… 105 BIBLIOGRAFÍA……………………………………………………………………….....… 109 INTRODUCCIÓN 8 Rapamycin”) regula la biogénesis del ribosoma a varios niveles (Figura 4): participando tanto en la síntesis de pre-rRNAs a través de la regulación de la RNA polimerasa I (RNAPI) y la RNA polimerasa III (RNAPIII), como en la transcripción de los mRNAs de las proteínas ribosómicas, factores de ensamblaje y snoRNAs mediante la RNA polimerasa II (RNAPII), y finalmente en el procesamiento de los rRNAs y en el proceso de traducción [25, 31]. Procesamiento de los rRNAs La etapa inicial de la biogénesis del ribosoma es la transcripción del DNA ribosómico (rDNA). En S. cerevisiae, el rDNA se localiza en el cromosoma XII y está formado por un alto número (aproximadamente 150) de repeticiones en tándem [32]. FIGURA 4. La ruta TOR regula la biogénesis del ribosoma a varios niveles. La ruta TOR controla la biogénesis del ribosoma: (I) a través de la RNAPI afectando a la transcripción del pre-rRNA 35S, (II) a través de la RNAPIII promoviendo la transcripción del precursor de 5S y algunos snoRNAs, (III) y a través de la RNAPII regulando la expresión de las proteínas ribosómicas y los factores de ensamblaje. Adaptado de [31]. 9 Cada repetición consta de un gen que genera un transcrito primario de unas 9.1 kb que codifica el precursor 35S de los rRNAs 5.8S, 18S y 25S y que es transcrito por la RNAPI. En sentido contrario, presenta un gen que genera el precursor del rRNA 5S y que es transcrito por la RNAPIII (Figura 5). No todas las repeticiones se encuentran activas simultáneamente, sino que el número de copias que se transcriben en cada momento depende de las necesidades celulares y el estado metabólico de las células [33]. Los pre-rRNAs nacientes sufren distintas modificaciones químicas [34] y son procesados mediante la participación de exo- y endonucleasas [35]. FIGURA 5. Organización del rDNA de S. cerevisiae. La levadura S. cerevisiae contiene múltiples copias de los genes ribosómicos, en el cromosoma XII, que se transcriben como un único precursor de unas 9.1 kb en repeticiones en tándem. En este precursor se encuentran el pre-rRNA 35S que tras procesarse da lugar a los rRNAs maduros 5.8S, 18S y 25S. Éstos están flanqueados por dos regiones espaciadoras externas (5’ETS y 3’ETS) y están separados por dos regiones espaciadoras internas (ITS1 separando 18S de 5.8S e ITS2 separando a 5.8S de 25S). El precursor rRNA 5S está separado de 35S por otras dos regiones espaciadora que no se transcriben (NTS). Con las letras P y T se indica la región promotora y terminadora del transcrito policistrónico. El procesamiento de los rRNAs ocurre en el contexto de grandes complejos ribonucleoproteicos denominados partículas pre-ribosómicas [29]. Estas partículas además de los pre-rRNAs contienen multitud de factores no-ribosómicos (snoRNAs y proteínas), y distintos grupos de proteínas ribosómicas más o menos establemente ensambladas. En la mayoría de los casos, la perdida de función de alguno de estos factores origina defectos en el procesamiento de los pre-rRNAs. Hasta la fecha, sólo para unos pocos de estos factores, se ha descrito un mecanismo de acción molecular preciso. En el resto de los casos, la función exacta de los factores sigue siendo desconocida. 10 El procesamiento del precursor 35S comienza con los cortes endonucleolíticos consecutivos en los sitios A0 y A1, localizados en el 5’ETS, y posteriormente en el sitio A2 localizado en la región interna ITS1; el corte en el sitio A2 da lugar a los precursores 20S y 27SA2, que pertenecen a rutas de maduración aparentemente independientes de las partículas pre-40S y pre-60S [36, 37] (Figura 6). Numerosas evidencias experimentales indican que la eliminación del 5’ETS del prerRNA 35S podría ocurrir antes de que éste último sea transcripto por completo [38], es decir, ocurrir de manera co-transcripcional. Estudios cinéticos de la maduración de los pre-rRNAs y la visualización mediante microscopia electrónica (EM) del transcrito naciente del prerRNA apoyan el hecho de que el corte en el sitio A2 pueda ocurrir co-transcripcionalmente. En S. cerevisiae se estima que aproximadamente un 70% de los pre-rRNAs se procesan cotranscripcionalmente en fase exponencial de crecimiento [39, 40]. En cualquier caso, como se ha mencionado anteriormente, el corte en el sitio A2 separa las rutas de maduración de partículas pre-40S y pre-60S, originando los precursores 20S y 27SA2 los cuales se procesan de manera aparentemente independiente. En el caso de la formación de la partícula pre-40S, el pre-rRNA 20S se procesa en su extremo 3’ en el sitio D por la endonucleasa Nob1 en el contexto de una partícula ribosómica denominada “80S_like” en el citoplasma [41]. En el caso del pre-rRNA 27SA2 el procesamiento es más complejo. El 27SA2 puede seguir dos rutas alternativas de maduración, cuya principal diferencia es la forma de procesar la región interna ITS1. En la ruta principal, aproximadamente, el 85% de todo el pre-rRNA 27SA2 se procesa en el sitio A3 por la endonucleasa RNasa MRP [42], resultando en el pre-rRNA 27SA3 que es digerido posteriormente 5’-3’ exonucleoliticamente hasta el sitio B1S para dar lugar al precursor 27SBS, que posee el extremo 5’ maduro del rRNA 5.8SS [43, 44]. En la ruta minoritaria, aproximadamente, el 15% de todo el pre-rRNA 27SA2 es procesado directamente en el sitio B1L, mediante un corte endonucleolítico por una nucleasa aún desconocida. Este corte origina el pre-rRNA 27SBL que presenta el extremo 5’ maduro del rRNA 5.8SL [45]. Al mismo tiempo que se completa el procesamiento del sitio B1, ocurre el procesamiento del extremo 3’ del rRNA 25S mediante la digestión 3’-5’ exonucleolítica desde el sitio B0 hasta el sitio B2 presente en el pre-rRNA 27SA [46, 47]. Parece ser que los pre-rRNAs 27SBS y 27SBL se procesan siguiendo las mismas reacciones de maduración. La primera reacción consiste en el corte en el sitio C2, que separa los pre-rRNAs 7S y 25.5S (26S) que darán lugar a los rRNAs maduros 5.8S y 25S, respectivamente [46]. Recientemente se ha demostrado in vitro que el “complejo Las1” 11 FIGURA 6. Esquema del procesamiento de los rRNAs en S. cerevisiae. (A) Organización de una repetición del rDNA donde se indica la localización de los sitios de corte. (B) Esquema de la ruta de maduración de los prerRNAs. En rojo se muestran las enzimas endonucleolíticas y en verde las enzimas exonucleolíticas. Las nucleasas aún no identificadas se han marcado con un símbolo de interrogación. Adaptado de [48]. Las1 complex Cleavage C2 Las1 complex Cleavage C2 12 compuesto por cuatro subunidades: Las1, una endonucleasa; Grc3, una 5’ polinucleótido kinasa; Rat1, una 5’-3’ exonucleasa y su cofactor Rai1, una 5’ pirofosfatasa, es el responsable del corte en el sitio C2 [49]. Este corte genera un pre-rRNA 7S con un extremo 3’ con un 2’,3’ fosfato cíclico, y un pre-rRNA 26S con un extremo 5’ con un grupo OH. El grupo fosfato cíclico que presenta el pre-rRNA 7S en su extremo 3’ podría ser el responsable de la estabilidad de este intermediario y protegerlo del procesamiento a 5.8S [49]. El pre-rRNA 7S se procesa en el extremo 3’ por una serie de 3’-5’exonucleasas [50-53] que da lugar al menos a dos intermediarios estables, los pre-rRNAs 5.8S+30 generado por la subunidad Rrp44 del exosoma, y el pre-rRNA 6S generado por la subunidad Rrp6. Recientemente, se ha demostrado que la maduración del extremo 3’ del precursor 6S ocurre en el citoplasma [54] mediante una serie de reacciones consecutivas llevadas a cabo por las nucleasas Rex1, Rex2, Rex3 [52] y Ngl2 [53]. Esto da lugar a las dos especies de rRNA 5.8S denominadas 5.8SL y 5.8SS. El pre-rRNA 26S generado tras el corte en C2 es fosforilado por Grc3 permitiendo que Rat1-Rai1 se procese a un intermediario 25S’; posteriormente, las enzimas Rat1/Rrp17 maduran este intermediario hasta la especie madura del rRNA 25S [44, 49, 55]. Por otra parte de manera independiente, el rRNA 5S se transcribe por la RNAPIII como un precursor que presenta su extremo 5’ maduro, pero cuyo extremo 3’ se extiende varios nucleótidos del extremo 3’ maduro. La maduración del pre-rRNA 5S consiste en la digestión 3’-5’ exonucleolítica de estos nucleótidos llevada a cabo por la exonucleasa Rex1 [52, 56]. Las proteínas ribosómicas en S. cerevisiae Las 79 proteínas ribosómicas de S. cerevisiae (Figura 7A y 7B), se codifican por 138 genes; en la mayoría de los casos, los genes están duplicados dando lugar a proteínas parálogas. Los mRNAs de las proteínas ribosómicas representan el 50% de toda la transcripción realizada por la RNAPII. Los genes de estas proteínas constituyen un agrupamiento funcional conocido como regulón RP [57]; la característica que presentan la mayoría de estos genes es la presencia en sus promotores de un sitio de unión del factor de transcripción Rap1 [58]. Además, algunos genes de proteínas ribosómicas presentan un sitio de unión para el factor Abf1 [25]. Se ha demostrado que la unión defectiva de Rap1 a los promotores de las proteínas ribosómicas provoca una disminución del 75% de la transcripción de estos genes [59]. En la regulación de la transcripción del regulón RP intervienen diversos factores de transcripción y señales intra- y extracelulares que están 13 FIGURA 7. Estructura tridimensional de las proteínas ribosómicas de la subunidad pequeña y grande del ribosoma de S. cerevisiae. (A) Estructura tridimensional de las 33 proteinas ribosómicas que conforman la subunidad pequeña del ribosoma de S. cerevisiae. (B) Estructura tridimensional de las 46 proteínas ribosómicas que conforman la subunidad grande del ribosoma de S. cerevisiae. Las proteínas ribosómicas aunque bien conservadas, presentan extensiones específicas en eucariotas, que juegan papeles importantes en diferentes puntos del proceso de ensamblaje de la propia proteína o interactuando con otras proteínas vecinas. La representación fue generada con el programa Chimera v.1.11.2 a partir del archivo pdb: 4V88. A A B 14 integradas en la ruta TOR (Figura 8), la cual como se ha mencionado anteriormente, mantiene el correcto balance en la producción de los rRNAs y las proteínas ribosómicas [25, 31]. FIGURA 8. Regulación de la transcripción de los genes de proteínas ribosómicas (RP). Determinadas señales tanto extracelulares como intracelulares inducen o reprimen la ruta TOR y RAS/PKA como se indica en la figura. Estas señales se canalizan mediante una cascada de señalización que implica factores integradores como Sfp1 y Sch9. El factor activador clave de los genes RP es Ifh1, el cual se une a los promotores gracias a su interacción con el factor Fhl1 [60]. Mrs6 es un factor importante implicado en la regulación de la localización subcelular de Sfp1, y éste a su vez, regula la localización de los factores Ifh1 y Fhl1 en el núcleo. Por otro lado la actividad quinasa del factor Sch9 es esencial; se especula que fosforila al factor Rap1 [61, 62]. Rap1 y Hmo1 son factores de transcripción que cooperan para regular positivamente la transcripción de los genes de las proteínas ribosómicas. Esa1 es una acetil-transferasa de histonas que elimina la acción represora de los nucleosomas. Hay dos sitios de unión para Rap1 en el promotor de la mayoría de los genes de proteínas ribosómicas. Cuando la proteína Rap1 se une, se activa la transcripción. Se ha observado que algunos genes de proteínas ribosómicas presentan un sitio de unión al factor Abf1 en sus promotores. Adaptada de la Tesis Doctoral de Fernando Gómez Herrero. 15 Las proteínas ribosómicas son normalmente de pequeño tamaño y de carácter básico, con la excepción de las proteínas del tallo P (P1 y P2) que aunque pequeñas presentan un carácter ácido; el carácter básico proporciona un entorno en el cual se neutraliza las cargas negativas de los residuos fosfatos de los rRNAs [63]. Muchas de las proteínas ribosómicas presentan una estructura diferenciada en dos dominios: una región globular fuertemente estructurada que en la mayoría de los casos se localiza en la superficie del ribosoma, y una región formada por una o varias extensiones normalmente intrínsecamente desordenadas que es frecuente que se introduzcan hacia el interior del ribosoma (Figura 9). FIGURA 9. Estructura tridimensional de la proteína ribosómica L3 (uL3) de S. cerevisiae. (A) Estructura tridimensional de L3 (uL3) donde se muestra una región coloreada en verde que forma los dominios globulares de la proteína y una región en rojo que corresponde con las extensiones de la misma, una amino-terminal y otra como un lazo central. (B) Localización de la proteína L3 (uL3) en el contexto de la subunidad 60S del ribosoma. Se observa que el dominio globular de la proteína se sitúa en la superficie del ribosoma en contacto con el exterior mientras que las extensiones de la proteína se introducen en el interior del ribosoma. Realizado con el programa Chimera v.1.11.2 a partir de los archivos pdb: 3u5i y 3u5h. Muchos genes de proteínas ribosómicas se encuentran duplicados, las proteínas parálogas pueden ser idénticas pero en numerosos ejemplos no lo son totalmente. Se ha llegado a especular que el ensamblaje de uno u otro parálogo afecta al funcionamiento del ribosoma. Desde un punto de vista general, tomando la posibilidad de diferentes especies de rRNA (el caso de 5.8SS o 5.8SL) y proteínas ribosómicas, podemos imaginar un escenario de ribosomas con distinta composición, que podría reflejarse en distinta funcionalidad, es decir, un escenario de ribosomas especializados. Esta idea se ha confirmado para ribosomas de distintos tejidos de organismos eucariotas superiores pero 16 hasta el momento no se ha podido demostrar selectividad real de estos ribosomas hacia ciertos mRNAs para que se traduzcan diferencialmente [64]. Durante mucho tiempo se ha pensado que la función ribosómica residía exclusivamente en las proteínas ribosómicas y que los rRNAs servían de esqueleto estructural. Con la aparición del concepto de RNA catalítico y la obtención de datos que relacionaban de manera casi exclusiva a los rRNAs con la función ribosómica, se estableció la hipótesis no probada de que el ribosoma es una ribozima, dónde la actividad catalítica del centro peptidil-transferasa es llevada a cabo por el rRNA 25S (o 23S) [65, 66]. En las últimas dos décadas, la resolución de estructuras de ribosomas de distintos organismos a nivel atómico [67] ha confirmado que las proteínas ribosómicas se encuentran relativamente lejos de los centros funcionales como para ser responsables de la acción catalítica, aunque son esenciales para el funcionamiento general del ribosoma [66]. En las últimas décadas casi todo el esfuerzo investigador se había enfocado en el análisis del papel de los factores de ensamblaje en la biogénesis del ribosoma. Poco se conocía sobre la importancia de las proteínas ribosómicas en este proceso [68-77]. Se han encontrado principios generales entre el ensamblaje de ribosomas procariotas y eucariotas, aunque debido a la mayor complejidad del ribosoma eucariota los procesos no son del todo equivalentes ni están aun completamente resueltos [78]. La nomenclatura de las proteínas ribosómicas ha sufrido cambios drásticos no deseables a lo largo de los últimos años, dando lugar a confusiones debido a que proteínas ortólogas procariotas y eucariotas tenían nombres distintos; así la proteína L5 bacteriana se corresponde con la proteína L11 de S. cerevisiae, a su vez la proteína L5 de levadura es la proteína L18 en bacterias. Esto parece haber sido solucionado con el establecimiento de una nomenclatura que evidentemente necesita un periodo de adaptación para su correcta asimilación por parte de la comunidad científica que estudia el ribosoma [79] (Tabla 1). Maduración de la partícula pre-ribosómica temprana 90S Como he mencionado anteriormente, el procesamiento de los rRNAs ocurre en el contexto de grandes complejos ribonucleoproteicos formados por distintos pre-rRNAs, algunas proteínas ribosómicas y numerosos factores de ensamblaje. La composición de las partículas pre-ribosómicas es distinta dependiendo del estado de maduración de las mismas. La primera partícula pre-ribosómica detectada es la partícula 90S o también conocida como procesoma SSU (del inglés Small-SUbunit processome) [80, 81]. La partícula 90S es un complejo macromolecular de aproximadamente 2.2 MDa que contiene al 17 TABLA 1. Nomenclatura unificada de las proteínas ribosómicas. Se muestra la nueva nomenclatura adaptada por el área de trabajo del ribosoma de todas las proteínas ribosómicas de las subunidad pequeña (izquierda) y de la subunidad grande (derecha). Se señalan cuales son exclusivas de bacterias (B), cuáles de arqueas (A), y cuáles de eucariotas (E). Adaptado de [79]. 24 FIGURA 14. Últimas reacciones de maduración de la subunidad 40S. Modelo de los eventos de maduración citoplasmática de la subunidad pequeña. En amarillo, se muestran los factores que se unen de forma estable a partículas pre-40S; en magenta, se muestran las proteínas ribosómicas, y en color verde, los factores que se unen de forma más débil. La partícula pre-40S se muestra en color gris claro y la subunidad 60S en gris oscuro; el mRNA se muestra en color azul. Los distintos pasos se describen previamente en el texto. Adaptado de [78]. Recientemente se ha cristalizado el factor Tsr1, una proteína estructuralmente parecida a una GTPasa traduccional pero que no presenta ni sitio de unión a GTP ni actividad hidrolítica. Se ha observado que Tsr1 ocupa una posición que solapa con la de la GTPasa eIF5B y la ATPasa Rio1. La asociación de Tsr1 a las partículas pre-40S bloquea el canal de unión de los mRNAs, previene la asociación de la misma con la subunidad 60S y bloquea la unión de los factores eIF5B y Rio1. El mecanismo por el que se libera el factor Tsr1 de la partícula pre-40S aún se desconoce [123]. El ensamblaje de la subunidad 60S El ensamblaje de la subunidad 60S presenta mayor complejidad que el de su análoga pequeña. A diferencia del ensamblaje de la subunidad pequeña que ocurre en su mayor parte de forma co-transcripcional, sólo las primeras etapas de la formación de las partículas pre-60S más tempranas ocurren de esta forma. Como he mencionado anteriormente, la maduración de la subunidad 60S se inicia con el corte en el sitio A2 del prerRNA 35S [39, 40, 124]. Una vez finalizada la transcripción se forma el primer precursor detectable de pre-rRNA denominado pre-rRNA 27SA2. El factor Rrp5 se localiza asociado en 25 el ITS1 adyacente a los sitios A2 y A3, y se ha demostrado que favorece los cortes en A0, A2 para producir el rRNA 18S y en el sitio A3 por la RNasa MRP para dar lugar a los rRNAs 5.8S y 25S [100]. Muchas de las proteínas ribosómicas de esta subunidad y numerosos factores de ensamblaje están involucrados específicamente en el proceso de maduración [95]. Las proteínas ribosómicas en esta etapa aún presentan interacciones débiles con los pre-rRNAs que en muchos casos no son las interacciones definitivas que tienen con los rRNAs maduros. A medida que transcurre el proceso de maduración, estas interacciones se van haciendo más robustas y las proteínas ribosómicas van ocupando sus posiciones definitivas [125]. En un principio, los estudios para comprobar el papel de las proteínas ribosómicas durante el ensamblaje de las subunidades 60S se enfocaban en provocar únicamente la represión transcripcional de unas pocas proteínas individuales, y la constatación fenotípica de los efectos tras esta represión en el crecimiento celular, en los niveles de subunidades totales, polirribosomas, procesamiento de los pre-rRNAs y el transporte núcleocitoplasmático de partículas pre-ribosómicas [126-137]. Actualmente, el estudio del papel de las proteínas ribosómicas también incluye análisis sistemáticos en los que se estudia la composición de partículas pre-ribosómicas purificadas y cómo éstas cambian tras la pérdida de función o eliminación de aquellas proteínas ribosómicas en estudio [125, 138-140]. Gracias a estos estudios, se ha descubierto qué conjunto de proteínas ribosómicas afectan específicamente a las etapas tempranas de la maduración de la subunidad 60S, cuales afectan a las etapas intermedias y cuales a las etapas tardías y citoplasmáticas. Curiosamente, los conjuntos de proteínas que afectan principalmente a etapas tempranas o tardías en la maduración de las partículas ribosómicas tienen una correlación con su localización en el ribosoma. Así, las proteínas necesarias para las etapas tempranas del procesamiento del pre-rRNA 27SA2 se localizan en la superficie soluble de la subunidad 60S unidas a los dominios I y II del rRNA 25S. El siguiente grupo de proteínas, que son necesarias para las etapas intermedias de maduración se encuentran localizadas en los alrededores de la salida del túnel de salida de los polipéptidos nacientes (PET, en inglés), unidas a los dominios I y III del rRNA 25S y al rRNA 5.8S. Por otro lado, las proteínas que se necesitan para el procesamiento del pre-rRNA 7S se localizan en la superficie de contacto entre subunidades. Por último, las proteínas necesarias para los últimos estadíos nucleares y citoplasmáticos de maduración se localizan alrededor de la protuberancia central, donde se sitúa el rRNA 5S, entre los dominios II y V del rRNA 25S (Figura 15). Este proceso jerárquico recuerda vagamente al proceso que sigue el ensamblaje in vitro de la subunidad grande bacteriana [141], sugiriendo que muchos de los principios que gobiernan el ensamblaje de la subunidad grande están como sucede para la subunidad pequeña 26 conservados evolutivamente, a pesar de la mayor complejidad de la biogénesis del ribosoma en eucariotas. Si desgranamos más específicamente las etapas de maduración de la subunidad 60S, llama la atención que durante las etapas tempranas de su ensamblaje es necesaria la estabilización de los extremos 3’ y 5’ del pre-rRNA 27SA2 la cual se lleva a cabo mediante la participación de una serie de factores entre ellos Rrp5 [100]. Se ha observado que la proteína ribosómica L3 (uL3) se une a los dominios del pre-rRNA 27SA2 donde se sitúan sus extremos. La proteína L3 (uL3) en el ribosoma maduro ocupa una posición cercana al extremo 5’ del rRNA 5.8S y al extremo 3’ del rRNA 25S [142]. Además de la proteína L3 (uL3), se ha observado que un conjunto de factores de ensamblaje relacionados genéticamente con L3 (uL3) participa en esta estabilización inicial y compactación de la partícula pre-60S más temprana. Estos factores incluyen, además de Rrp5, las proteínas nucleolares Npa1/Urb1, Npa2/Urb2, Rsa3, Nop8 y las helicasas de RNA Dbp6, Dbp7, Dbp9 [100, 143]. Transcurridos estos primeros pasos de maduración, en los pasos intermedios se engloban los procesos de eliminación de la región interna ITS1 para lo cual es necesario la formación de una estructura a modo de pseudonudo en la estructura del rRNA 5.8S y el plegamiento del ITS2. Posteriormente ocurre el plegamiento de los dominios I y II creando una plataforma que ayuda a la formación de la estructura terciaria de los rRNAs [144]. El siguiente paso en el ensamblaje es la asociación de proteínas ribosómicas con los dominios I y III, permitiendo el corte en el sitio C2 en la región interna ITS2. Los factores necesarios para el corte en el sitio C2 (Nsa2 y Nog2/Nug2) se reclutan gracias al ensamblaje de algunas proteínas ribosómicas (L9 (uL6), L17 (uL22), L19 (eL19), L23 (uL14), L25 (uL23), L26 (uL24), L27 (eL27), L31 (eL31), L34 (eL34), L35 (uL29), L37 (eL37) [145, 146]. Por otro lado estas proteínas ribosómicas asociadas en estos pasos intermedios son necesarias para el correcto ensamblaje posterior de las proteínas ribosómicas L2 (uL2), L39 (eL39) y L43 (eL43) localizadas en la superficie inter-subunidad adyacentes al centro peptidil-transferasa (PTC, en inglés) [125]. A su vez las proteínas L2 (uL2) y L43 (eL43) junto con Nog2 son necesarias para el procesamiento del pre-rRNA 7S [125]. Mencionar que no se conoce el mecanismo por el cual se acopla el ensamblaje de estas proteínas con el procesamiento de este pre-rRNA. Recientemente se ha purificado un intermediario de pre-60S utilizando el factor Nog2 etiquetado con TAP, donde se ha observado que la GTPasa Nog2 está presente durante tres procesos importantes de la maduración de subunidades 60S: la rotación del complejo 5S-RNP, la construcción del PTC y la eliminación de ITS2. En este intermediario 27 A) B) FIGURA 15. Ensamblaje de las proteínas ribosómicas de la subunidad grande del ribosoma. A) Representación de la estructura secundaria de los rRNAs de la subunidad 60S. B) Representación tridimensional del rRNAs de la subunidad 60S; en amarillo, se muestran las proteínas que se ensamblan de forma temprana las cuales se localizan en la superficie de la subunidad 60S. En azul, aquellas proteínas que se ensamblan de manera intermedia y que se localizan alrededor de la salida del túnel de salida del polipéptido naciente. Por último, en rojo, se muestran las proteínas ribosómicas que se ensamblan de forma tardía y que se encuentran formando parte de la protuberancia central. Adaptada de [78]. se han observado catorce factores asociados a una región que han denominado región “arc” que se localiza en la superficie de interacción con la subunidad pequeña, entre la protuberancia central y el túnel de salida del polipéptido. Estos factores son Rsa4, Nsa2, 28 Mtr4, Nog1, Tif6, Rlp24, Bud20, Arx1, Nog2, Rpf2 y Rrs1 y cinco factores adyacentes a la zona de ITS2, Nop53, Nop7, Rlp7, Cic1, Nop15 [147]. La actividad de la ATPasa Rea1 provoca cambios conformacionales en las partículas pre-60S nucleoplásmicas tardías que estimulan la actividad GTPasa de Nog2, cuyas actividades son necesarias para el transporte de éstas al citoplasma [148]. La liberación de Nog2 permite la asociación del factor de transporte núcleo-citoplasmático Nmd3 cuyo sitio de unión solapa con el de Nog2 [148, 149]. Por otro lado, la GTPasa Nog1 presenta una función dual, su dominio amino terminal interacciona con los factores Nog2 y Nsa2 mientras que su dominio carboxilo terminal interacciona con los factores Tif6, Rlp24, Arx1 y las proteínas ribosómicas L3 (uL3), L31 (eL31), L22 (eL22), L19 (eL19) y L35 (uL29). Se sugiere que mientras el extremo amino de Nog1 actúa en el remodelado del centro, el extremo carboxilo terminal aparentemente actúa como un “andamio molecular” para la asociación de numerosos factores de ensamblaje y el ensamblaje de algunas proteínas ribosómicas además de participar en un control de calidad de la construcción del túnel de salida del polipéptido [147, 150]. Como se mencionó anteriormente en este intermediario se observó que Nop53 se localiza cerca del sitio ITS2, lo cual tiene sentido porque Nop53 recluta el factor Mtr4, el cual participa en la eliminación de ITS2 mediante el exosoma [151]. El último paso antes de la salida de partículas pre-60S del núcleo es la formación de la protuberancia central que contiene el rRNA 5S unido a las proteínas L5 (uL18) y L11 (uL5). Recientemente, ha sido demostrado que las proteínas L5 (uL18) y L11 (uL5) se importan al núcleo juntas a través de un complejo formado por la carioferina Kap104 y la importina Syo1. Los extremos aminos de las proteínas L5 (uL18) y L11 (uL5) interaccionan con Syo1, cuya secuencia de señalización nuclear NLS es reconocida por Kap104. Tras el transporte al núcleo, la proteína Ran unida a GTP libera a Kap104 y el complejo Syo1-L5- L11 se asocia con el rRNA 5S. Syo1 se libera probablemente durante el ensamblaje del 5S- RNP al pre-60S [152] (Figura 16). El complejo 5S-RNP se ensambla en la partícula preribosómica 60S mediante los factores de ensamblajes Rpf2 y Rrs1 [130]. En este paso, actúan los factores Rsa4 y Nog1 que contribuyen en la estabilización de la protuberancia central prematura y el remodelado del 5S-RNP que inicialmente presenta una conformación rotada 180º de su posición final, ocasionando a su vez, la rotación de algunas hélices del rRNA 25S [153]. Aparentemente esta conformación alterada proporciona los sitios de unión de factores de ensamblaje durante momentos específicos del proceso de maduración y establece un control temporal de la formación de la protuberancia central. La proteína ribosómica L21 (eL21) se localiza en la protuberancia central cerca del complejo 5S-RNP. Esta proteína se ha demostrado que es necesaria para la interacción de factores implicados en el remodelado de la protuberancia central como son Rsa4 y Nog1 29 mencionados anteriormente. Por otro lado, el ensamblaje de la proteína L21 (eL21) parece ser un pre-requisito para que la ATPasa Rea1 pueda liberar de manera eficiente a los factores Rsa4 y Ipi1 [125, 154]. La liberación de los factores Rpf2 y Rrs1 unidos con el 5S- RNP, y del factor Rsa4 son necesarios para la correcta rotación del 5S-RNP a su posición final. La reorganización del 5S-RNP y la liberación del factor Nog2 parecen estar acoplados al transporte de la partícula pre-60S al citoplasma, la liberación del factor Nog2 permite que el factor de transporte núcleo-citoplasmático Nmd3 se asocie a las partículas pre-60S, puesto que ocupan posiciones solapantes [148]. Por otro lado parece ser que la reorientación de la protuberancia central favorece la asociación del factor Mex67 para llevar a cabo el transporte al citoplasma [155]. FIGURA 16. Ensamblaje del complejo 5S-RNP. El complejo Syo1-L5-L11 que se forma en el citoplasma, se importa al núcleo con la ayuda de la carioferina Kap104. En el núcleo, el complejo Syo1-L5-L11 se unen al rRNA 5S. La actividad de RanGTP unida a Kap104 promueve la liberación de Syo1. En este punto, el complejo 5S- RNP se incorpora a la partícula pre-ribosómica 60S. Los factores Rpf2 y Rrs1 ayudan en el reclutamiento de 5S- RNP a la partícula pre-60S generando la protuberancia central prematura. Adaptada de [156]. Una vez en el citoplasma, las partículas pre-60S se someten a los últimos pasos de maduración, entre ellos el procesamiento del pre-rRNA 6S a 5.8S, la asociación de las últimas proteínas ribosómicas y la liberación de los factores de ensamblaje que aún quedan unidos (Figura 17). Las proteínas ribosómicas de ensamblaje citoplasmático se localizan en la superficie de la subunidad que interacciona con la subunidad pequeña, adyacentes a la protuberancia central, al centro peptidil-transferasa y al centro activador de GTPasa. Se 30 piensa que los últimos pasos de maduración están precisamente destinados a completar los sitios activos. Se ha demostrado que la ausencia de las proteínas ribosómicas que se ensamblan en estos últimos pasos citoplasmáticos, como las proteínas L29 (eL29) y L40 (eL40), o la represión transcripcional de la proteína L10 (uL16), dan lugar a defectos en la unión de la subunidad grande con la subunidad pequeña durante el proceso de traducción [134, 157, 158]. El ensamblaje prematuro de proteínas como L10 (uL16), L24 (eL24) o P0 (uL10) en partículas pre-60S está bloqueado estéricamente por factores de ensamblajes que ocupan sus sitios de unión, constatando nuevamente la presencia de factores tipo “placeholders”. Es durante los últimos pasos de maduración citoplasmática cuando las subunidades 60S nacientes se someten a un nuevo chequeo para comprobar el ensamblaje correcto del sitio P en los centros peptidil-transferasa y activador de GTPasa. El primer factor en actuar parece ser que es la ATPasa AAA Drg1, la cual es reclutada y activada por el factor Rlp24; a su vez Drg1 promueve la liberación de Rlp24, permitiendo el ensamblaje posterior de la proteína ribosómica L24 (eL24) que es paráloga al factor [159, 160]. Esta etapa permite la unión del factor Rei1 que junto con la ATPasa Ssa1 y su cofactor Jjj1 liberan los factores Arx1 y Alb1 [161-163]. Por otro lado, la proteína ribosómica L12 (uL11) recluta al factor de ensamblaje Yvh1 que interviene en la liberación de Mtr4 permitiendo que la proteína ribosómica P0 se ensamble de forma irreversible a la partícula pre-60S citoplasmática [161, 164-166]. Muy recientemente, se ha demostrado que la liberación de Mtr4 favorecida por el factor Yvh1 permite la asociación de los factores de transporte Mex67-Mtr2 en el núcleo en el sitio que ocupaba Mtr4, permitiendo su transporte al citoplasma. Allí, los factores Yvh1 y Mex67-Mtr2 se liberan y favorecen el ensamblaje de la proteína ribosómica P0 [167]. Sin embargo, en ausencia de la proteína ribosómica L12 (uL11), que no es esencial, no se ha observado que haya diferencias en la eficiencia de reciclaje del factor Mtr4 al núcleo [168]. La proteína ribosómica L10 (uL16) presenta un lazo flexible en su estructura que, debido a su proximidad al sitio P, es responsable de detectar si el ensamblaje del centro peptidiltransferasa es correcto. En ese caso, se activan los factores Efl1 y Sdo1, que favorecen la liberación del factor Tif6 [169]. A su vez, este proceso favorece la liberación del factor Nmd3 llevada a cabo por la GTPasa Lsg1. Es el correcto ensamblaje del tallo P el que activa y une el factor Efl1, el cual podría acoplar este punto de control con la liberación de factores que previenen la interacción de la subunidad grande con la subunidad pequeña de forma prematura. 31 Mecanismos de vigilancia en la biogénesis del ribosoma Como he estado mencionando en secciones anteriores, los puntos de verificación estructural y funcional de las partículas pre-ribosómicas son frecuentes a lo largo de la ruta de maduración de las subunidades ribosómicas. Éstos aseguran el correcto ensamblaje del ribosoma. Muchos de estos controles ocurren en el nucléolo. Entre ellos destacan las distintas verificaciones realizadas por el complejo TRAMP asociado al exosoma [170]. Esta ruta parece actuar en los casos en los que ciertos factores de ensamblaje no son capaces de unirse o llevar a cabo su función, lo que podría provocar el bloqueo de las partículas pre-ribosómicas que no pueden progresar en su maduración, simplemente por falta de reciclado de los factores asociados. FIGURA 17. Últimas etapas de la maduración de la subunidad 60S. Las partículas pre-60S citoplasmáticas contienen solo unos pocos factores de ensamblaje que se liberan tras las últimas reacciones de remodelado citoplasmático. La incorporación de L29 (eL29) en el citoplasma requiere evidencia experimental. Es de destacar la liberación del factor Tif6 que provoca la activación de la GTPasa Lsg1, que a su vez promueve la liberación del factor de exportación Nmd3. Por otro lado, el ensamblaje de la proteína L10 (uL16) requiere de la participación de la chaperona Sqt1, la cual es también necesaria para la liberación del factor Nmd3. Las proteínas acídicas P1 y P2, que pueden existir en su forma libre, se asocian aparentemente de manera co-traduccional a las subunidades 60S durante la fase de iniciación de la traducción. Adaptada de [78]. El complejo TRAMP está compuesto por una polimerasa de poli-adenina (Tfr4 ó Tfr5), una helicasa de RNA (Mtr4) y una proteína con dedos de zinc (Air1 ó Air2). Este complejo se encarga de añadir pequeñas colas de poli-(A)n en el extremo 3’ de los RNAs aberrantes, incluyendo aquellos que provienen de los rRNAs. Posteriormente, el exosoma 32 detecta la cola de poli-(A)n y realiza la degradación en sentido 3’-5’. Se ha sugerido que la mayoría de los complejos TRAMP-exosoma se concentran en unos compartimentos nucleolares denominados “No-body” [170]. En el citoplasma, las partículas pre-ribosómicas y los ribosomas aberrantes son degradados mediante la ruta denominada “Non-functional rRNA Decay” (NRD). Según se trate de una subunidad pequeña o una grande aberrante, esta ruta utiliza distintos factores. Así, el NRD de la subunidad pequeña comparte componentes con una ruta de degradación de mRNAs denominada “No-Go Decay” (NGD) [171], mientras que el NRD de la subunidad grande utiliza la ubicuitinación [172]. Mientras que la degradación de la subunidad pequeña parece ser dependiente de traducción [171], la degradación de la subunidad grande se lleva a cabo por el proteasoma citoplasmático (UPS en inglés) y es dependiente de ubicuitinación [172]. Los sustratos de degradación de la subunidad pequeña al igual que la de los mRNAs se localizan en los gránulos citoplasmáticos, conocidos como “P-bodies”, donde se encuentran las enzimas encargadas de la degradación, tales como la exonucleasa 5’-3’ Xrn1, y las GTPasas Ski7 y Hbs1. En cambio, los sustratos de degradación de la subunidad grande se acumulan en compartimentos peri-nucleares [172]. Finalmente, mencionar que dependiendo del estado fisiológico (condiciones de estrés, inanición, o fase estacionaria, etc), las células activan mecanismos de reciclaje del exceso de ribosomas y partículas pre-ribosómicas tales como la ribofagia, un tipo de macroautofagia selectiva de subunidades 60S maduras. Esta ruta implica la proteasa de ubicuitina Ubp3, su activador Bre5 des-ubicuitinando y la ubicuitin-ligasa Rsp5 ubicuitinando ciertas proteínas asociadas a ribosomas [170, 173]. Se desconocen los mecanismos de ribofagia de subunidades pequeñas. La ruta ribofágica especifica de partículas pre-ribosómicas se denomina “Piecemeal Microautofagia of the Nucleus” (PMN), es un tipo de micro-autofagia selectiva donde se encuentra implicada la vacuola y la envuelta nuclear [174] (Figura 18). 33 FIGURA 18. Rutas principales de degradación del RNA ribosómico. (i) Mecanismo de degradación de los pre-rRNAs defectuosos por el complejo TRAMP-exosoma en el núcleo y nucléolo. (ii) Mecanismo de degradación de subunidades 40S maduras (18S NRD) llevaba a cabo por la maquinaria del NGD. (iii) Mecanismo de degradación de la subunidad 60S madura (25S NRD), en la que participa el proteasoma. (iv) Ribofagia de la subunidad 60S madura. (v) Micro-autofagia selectiva de componentes nucleolares (PMN). Adaptada de [170]. 41 Nuestro laboratorio tiene como objetivo principal el estudio del proceso de biogénesis del ribosoma eucariota y trabaja principalmente con Saccharomyces cerevisiae como organismo modelo. Entre otros proyectos, nuestro laboratorio intenta comprender la función precisa de las proteínas ribosómicas durante el ensamblaje de las subunidades ribosómicas. Así, en el mismo, se ha abordado la caracterización funcional de distintas proteínas ribosómicas tales como L3 [129], L35 [131], L26 [133], L37 [136], L40 [134] o P0 [166] de la subunidad grande y S31 [107], S18, S10 y S12 (en progreso) de la subunidad pequeña. Entre otras cosas por su proximidad con la proteína L3 en la subunidad ribosómica 60S madura, se planteó como objetivo de esta Tesis Doctoral la caracterización funcional de las proteínas ribosómicas L14 y L16, que como se explica a continuación son vecinas próximas entre sí en la subunidad 60S madura. Ambas proteínas presentan un tamaño pequeño y carácter básico, características generales de las proteínas ribosómicas. En la Figura 1 de este capítulo se muestra la localización de estas dos proteínas en el contexto ribosómico. FIGURA 1. Localización de L3, L14 y L16 en la subunidad grande del ribosoma de S. cerevisiae. Estructura tridimensional de la subunidad 60S (superficie de interacción entre subunidades). En verde, se muestra la proteína ribosómica L3, en azul la proteína L16 y en rojo la proteína L14. En celeste se muestran las restantes proteínas ribosómicas que componen la subunidad grande del ribosoma y en gris los rRNAs. Realizado con el programa Chimera v.1.11.2 a partir de los archivos pdb: 3u5h y 3u5i. A su vez, las proteínas L14 y L16 se encuentran cercanas a las proteínas L6 (eL6), L9 (uL16), L20 (eL20) y L33 (eL33) localizadas en la zona expuesta al solvente del ribosoma. Ambas muestran interacciones con los dominios II y VI del rRNA 25S (Ver Figura 15 de la introducción, pagina 32). Además L16 interacciona con el rRNA 5.8S (Figura 2). 42 La proteína L14 está codificada por dos genes parálogos, RPL14A y RPL14B. Estos dos genes muestran una similitud de secuencia muy alta que origina proteínas muy similares de 138 aminoácidos cada una; la única diferencia existente entre L14A y L14B consiste en un cambio del aminoácido alanina (A) en RPL14A, por una glicina (G) en RPL14B, en la posición 89. Las diferencias más llamativas se observan en regiones no codificantes tales como las presentes en el único intrón que ambos genes poseen. Este hecho sugiere que a diferencia de las regiones codificantes no ha habido restricción por presión selectiva en los intrones y abre la posibilidad de que puedan existir procesos de regulación específicos para cada parálogo. FIGURA 2. Interacción de las proteínas L14 y L16 con los rRNAs en el contexto de la subunidad 60S madura. Estructura tridimensional de la subunidad 60S del ribosoma de S. cerevisiae. Se muestran únicamente los rRNAs y las proteínas L14 (rojo) y L16 (azul). En color turquesa, se muestra el dominio II del rRNA 25S, y en color fucsia el dominio VI. En gris se muestra el resto de dominios del rRNA 25S, 5S y 5.8S. Realizado con el programa Chimera v.1.11.2 a partir de los archivos pdb: 3u5h y 3u5i. A su vez, la proteína L16 está también codificada por dos genes parálogos RPL16A y RPL16B. Ambos genes presentan una similitud de secuencia muy alta, sintetizando cada uno, una proteína de 198 aminoácidos. Las proteínas L16A y L16B presentan una similitud del 92% a nivel de aminoácidos. La mayoría de las diferencias de secuencias se encuentran nuevamente en el intrón que presentan ambos genes. Respecto a sus estructuras proteicas, L14 presenta un dominio globular y 2 extensiones, una mayor que corresponde con el extremo carboxilo terminal y una pequeña que corresponde con el dominio amino terminal (Figura 3). En el caso de la proteína L16, 43 ésta presenta un dominio globular y una extensión carboxilo terminal que se muestra en la Figura 3. FIGURA 3. Morfología de las proteínas L14 y L16. (A) Estructura tridimensional de la proteína ribosómica L14. Se marca con N y C sus extremos amino y carboxilo, respectivamente. (B) Estructura tridimensional de la proteína ribosómica L16. Se marca con N y C sus extremos amino y carboxilo, respectivamente. Realizado con el programa Chimera v.1.11.2 a partir del archivo pdb 3u5i. FIGURA 4. Interacción física entre L14 y L16. (A) Se muestra en turquesa y fucsia los dominios II y VI del rRNA 25S respectivamente, que conforman el entorno molecular de las proteínas L14 (rojo) y L16 (azul) interaccionando entre sí. (B) Detalle de la interacción de las proteínas L14 (rojo) y L16 (azul) en distintas orientaciones. Realizado con el programa Chimera v.1.11.2 a partir de los archivos pdb: 3u5h y 3u5i. B 44 Ambas proteínas interaccionan íntimamente entre sí por las α-hélices de sus extensiones C-terminales. Los aminoácidos implicados de la proteína L14 son los últimos 30 y de la proteína L16, los últimos 25. Ambas proteínas interaccionan principalmente mediante interacciones apolares entre residuos, exceptuando dos puentes salinos encontrados, uno entre el grupo carboxilo de la última alanina de la proteína L14 y el grupo ε-amino de la lisina 177 de L16, y el otro entre el grupo carboxilo de la última tirosina de L16 y el grupo guanidina de la arginina 109 de L14, según se deduce de la disposición de ambas proteínas en el cristal resuelto de la subunidad 60S madura (Figura 4). Como se ha mencionado anteriormente, uno de los objetivos de esta Tesis ha consistido en el estudio de las consecuencias funcionales de la disrupción de estas interacciones sobre el proceso de ensamblaje de las subunidades 60S. Al comienzo de este trabajo, la única información disponible sobre la proteína L16 era un estudio sistemático realizado por el grupo del profesor Philipp Milkereit. En este estudio, se reprimía la transcripción de algunos genes de proteínas ribosómicas y a continuación se estudiaba los efectos que éstos causaban sobre el procesamiento de los pre-rRNAs y el transporte nucleo-citoplásmatico de partículas pre-60S [175]. Dependiendo del parecido en los defectos observados tras la represión, las proteínas ribosómicas se clasificaron en distintos grupos funcionales. La proteína L16 junto a las proteínas ribosómicas L3, L4, L7, L8, L18, L20, L32 y L33 fue incluida en el grupo de proteínas cuya represión causaba una acumulación en el precursor 35S, no presentaban el precursor 27SA2 y afectaban al procesamiento del extremo 5’ del rRNA 5.8S. En esta Tesis quisimos profundizar en la caracterización funcional de la proteína L16 y su extremo carboxilo terminal en la síntesis del ribosoma. Respecto a la proteína L14, al comienzo de esta Tesis, no se disponía de ninguna información al respecto. En esta Tesis hemos iniciado el estudio funcional de la misma tras su represión transcripcional. También hemos estudiado la composición de partículas tempranas pre-60S tras la represión transcripcional de L14. Cuando este trabajo estaba en progreso, se publicó un artículo donde descubrían la interacción genética de la helicasa de RNA Mak5 y el factor de biogénesis Ebp2 con la proteína L14A. En este estudio, se propuso que posiblemente lo que ellos denominaron “grupo Mak5” (compuesto por Ebp2, Nop16 y Rpf1) podría estar implicado en dirigir los cambios conformacionales necesarios para el ensamblaje de un módulo preformado compuesto por las proteínas ribosómicas L6, L14 y L16 en el contexto de los segmentos de expansión ES7L y ES39L del rRNA 25S [176]. CAPÍTULO 1 Role of the yeast ribosomal protein L16 in ribosome biogenesis 47 INTRODUCTION Ribosomes are highly complex ubiquitous macromolecular assemblies that are responsible for the synthesis of all cellular proteins from mRNA templates. In all cells, ribosomes are composed of two ribosomal subunits (r-subunits), the large one about twice the size of the small one. The recent determination of the atomic structures of the r-subunits has confirmed that the majority of the ribosomal RNA (rRNA) and ribosomal proteins (rproteins) from prokaryotic ribosomes is conserved in eukaryotes, although additional rRNA sequences, referred to as expansion-segments, extra extensions within conserved r-proteins and eukaryote-specific r-proteins have increased significantly the mass and complexity of cytoplasmic eukaryotic ribosomes [22, 142, 177]. Synthesis of eukaryotic ribosomes is a highly complex process that takes successively place in the nucleolus, nucleoplasm and cytoplasm [30, 178, 179]. The speed, directionality and accuracy of this process depend on a myriad of small nucleolar RNAs and protein trans-acting factors [29, 106]. Within the nucleolus, the mature 18S, 5.8S and 25S rRNAs are first co-transcribed as a single large precursor by RNA polymerase I, whereas the pre-5S pre-rRNA is independently transcribed by RNA polymerase III [32, 180]. Pre-rRNAs are processed via well-known pathways that require a series of sequential endo- and exonucleolytic reactions [35, 48, 181]. Pre-rRNA processing takes place inside pre-ribosomal particles, which are formed by the association of many trans-acting factors with the prerRNAs and the assembled r-proteins; this process occurs concomitantly to most pre-rRNA modification and folding reactions [178]. Cleavage at processing site A2 leads to the formation of 43S and 66S pre-ribosomal particle intermediates of the small and large rsubunits, respectively, which then follow independent maturation pathways [179, 182]. While 43S pre-ribosomal particles are rapidly exported to the cytoplasm [183], early 66S preribosomal particles undergo hierarchical maturation involving pre-rRNA processing, association and release of trans-acting factors, structural remodelling of the particles, stable assembly of r-proteins and acquisition of export competence [29, 93, 179]. Upon appearance in the cytoplasm, the final maturation steps, which include dimethylation and cleavage of 20S pre-rRNA to yield mature 18S rRNA within the 40S r-subunit, 3'-end exonucleolytic processing of 6S pre-rRNA to mature 5.8S rRNA within the 60S r-subunit, and the dissociation of trans-acting factors and the assembly of the few remaining r-proteins in both r-subunits, enable r-subunits to acquire their competence in translation [179, 184]. Most of our current knowledge concerning ribosome biogenesis in eukaryotes comes from studies with the yeast Saccharomyces cerevisiae (hereafter named only yeast). More than 300 protein trans-acting factors have been identified and functionally characterized in yeast, although the precise functions by which these proteins operate are only beginning to 48 be unravelled [30, 178, 185]. As for trans-acting factors, functional analyses have demonstrated that many r-proteins act as active players in the maturation and nucleocytoplasmic transport of pre-ribosomal particles [144]. The principles governing the assembly of r-proteins start to be understood [76, 77, 125, 140, 144, 175], however, how r-proteins participate in driving formation or rearrangement of structures within pre-ribosomal particles remains in many cases yet to be unveiled. Finally, the functions of some r-proteins in ribosome biogenesis, especially those belonging to the large r-subunit, and their correct timing of assembly, are currently under intensive investigation. We are interested in understanding the contribution of 60S r-proteins to ribosome biogenesis (e.g. [129, 133, 136, 144, 166, 186]). In this study, we have undertaken the functional characterization of yeast L16 (uL13 according to the recently proposed r-protein nomenclature [79]). Yeast L16, which is encoded by two paralogous genes, RPL16A and RPL16B, shares a notable sequence identity with bacterial L13, which is an essential protein required for early assembly of 50S r-subunits in vivo [187, 188] and in vitro [74]. L16 also shows substantial homology to mammalian L13a, which is an early assembling 60S r-protein with no apparent role in pre-rRNA processing and the assembly and function of ribosomes, but with an important role in global rRNA methylation and, as an extra-ribosomal free rprotein, in modulating the translation of a subset of proteins in different cell types of the immune system [189-191]. Eukaryotic L16/L13a contains a specific C-terminal extension of unclear role during mammalian ribosome biogenesis [190]. In contrast to mammalian L13a and as previously reported in a systematic study [192], we show in this study that L16 is required for the normal accumulation of 60S r-subunits in yeast. Depletion of L16 causes instability of early and intermediate pre-ribosomal particles, as shown by low steady-state levels and massive turnover of the 27S and 7S pre-rRNAs. As a consequence, depletion of L16 impairs nucleocytoplasmic export of pre-60S r-particles. Moreover, L16 stably assembles into 90S pre-ribosomal particles. Finally, we clearly show that deletion of the eukaryote-specific C-terminal extension of yeast L16 causes lethality and impairs ribosome biogenesis; however, presence of this extension is not strictly essential for pre-rRNA processing and L16 assembly. While the present work was in progress, Milkereit and coworkers have independently reported similar findings on the role of the C-terminal extension of yeast L16 r-protein [192]. 49 RESULTS Depletion of L16 leads to a strong 60S r-subunit shortage The r-protein L16 protein is a constituent of the 60S r-subunit and it consists of an evolutionary conserved N-terminal globular body and a long eukaryote-specific C-terminal extension formed by two α-helices. L16 is located on the solvent-side of the 60S subunit in close proximity to L3, L14 and L33 (Fig. 1A). The globular domain of L16 binds to rRNA nucleotides within domains II and VI of 25S/5.8S rRNAs while its extension binds only nucleotides within domain VI, most of them in the expansion segment ES39L [11]. At the protein level, L16 mediates interactions with distinct regions of other r-proteins such as L6, L9, L20, L33, and most notably the most distal α-helix of its C-terminal extension interacts with the most distal α-helix of the eukaryote-specific C-terminal extension of L14 (Fig. 1B). Fig. 1. Location of L16 in the yeast 60S r-subunit. (A) Position of L16 and its immediate nearby r-proteins, in the context of the 60S r-subunit. The remaining 60S r-proteins are coloured in black, 25S and 5S rRNAs in pale gray, and 5.8S rRNA in red. Note that the 5' end of 5.8S rRNA is in proximity of the above r-proteins. (B) Structure of the ribosome-bound L16 and L14 r-proteins showing the interaction between their eukaryote-specific C-terminal extensions. The amino acids used for the serial deletion mutants of RPL16B are indicated. The representations were rendered with UCSF Chimera [193], using the atomic model for the crystal structure of the yeast 80S ribosome (PDB files 3U5I and 3U5H, [10]. A180 A168 K147 56 The eukaryote-specific C-terminal extension of L16 is required for growth Extensions of eukaryotic r-proteins have not yet thoroughly studied (i.e. see, [176, 201, 202]). To study the role of the eukaryote-specific C-terminal extension of L16 in cell growth and ribosome biogenesis, we cloned full-length RPL16B and three serial rpl16b truncation alleles under the control of the cognate RPL16B promoter into the YCplac111 centromeric plasmid. Upon transformation of these constructs into the rpl16a∆ rpl16b∆ [YCplac33-RPL16B] strain (hereafter RPL16 shuffle strain), cells were subjected to plasmid shuffling on 5-fluorootic acid (5-FOA) containing plates. Expression of wild-type L16B (fulllength L16B consists of 198 amino acids) or the truncated L16B protein variant lacking the last 18 amino acids (L16B-N180 construct), as the sole sources of L16 in the cells, conferred a wild-type growth phenotype (Fig. 7A) and a normal polysome profile (data not shown). However, expression of the truncated protein variants lacking the last 30 (L16B-N168 construct) or the last 51 amino acids (L16B-N147 construct) did not support growth (Fig. 7A). To further analyze the role of the C-terminal extension of L16, we transformed the above plasmid-borne truncated alleles of RPL16B into a yeast strain conditionally expressing an N-terminally HA-tagged L16B fusion-protein from a GAL1 promoter (GAL::HA-RPL16 strain). None of the truncated L16 variants conferred a dominant-negative growth phenotype on selective SGal medium (Fig. 7B). Upon depletion of wild-type HA-L16 on selective SD medium, only full-length L16B and the L16B-N180 variant supported growth, whereas the L16B-N168 and L16B-N147 proteins did not (Fig. 7B). Moreover, on selective SD medium, polysome profiles were normal upon expression of full-length L16B and the L16B-N180 variant, but indistinguishable of those obtained upon L16-depletion upon expression of the L16B-N168 and L16B-N147 proteins (data not shown). Next, we studied the expression and stability of the truncated forms of L16 from extracts of GAL::HA-RPL16 cells grown in selective SGal medium or shifted to selective SD medium for 12 h. As shown in Fig. 7C, all truncated forms of L16B were detected, although the L16B-N168 and L16B-N147 seemed to accumulate to a lesser extent; however, we cannot exclude the possibility that a substantial number of epitopes could have been removed with the truncations, and hence, that the recognition efficiency of the anti-L16 polyclonal antibodies have diminished resulting in weaker detection of the truncated L16B-N168 and L16B-N147 proteins. Taking into account that r-proteins unable to assemble into ribosomes are rapidly degraded (i.e. [203]), these results suggest that all the truncated forms of L16 could get incorporated into pre-60S rparticles (see below). It has been reported that the expression of certain r-protein gene mutants from plasmids leads to a much softer phenotype than their expression from their chromosomal loci (i.e. [204]). Indeed, for most duplicated r-protein genes, plasmidic 57 Fig. 7. Growth phenotype and expression levels of L16 in strains bearing wild-type or truncated variants of L16B. (A) In vivo phenotypes of the different C-terminal truncation mutants. Empty vector (YCplac111) and YCplac111-borne wild-type RPL16B or the indicated rpl16b alleles expressed from the RPL16B cognate promoter were transformed into the RPL16 shuffle strain, FEY251 [YCplac33-RPL16B]. Transformants were serially diluted 5-fold on SD-Leu and SD+5-FOA plates, which were incubated for 3 days at 30 ºC. The W303-1A strain (Wild type) was used as a control. (B) The strain FEY251 [pAS25-RPL16B], which expressed a 2xHA-tagged L16 fusion protein from the GAL1 promoter, was transformed with YCplac111-borne wild-type RPL16B or the indicated rpl16b alleles. Transformants were serially diluted 5-fold on SGal-Leu and SD-Leu plates, which were incubated for 3 days at 30 ºC. (C) Expression levels of YCplac111-borne wild-type RPL16B and the indicated rpl16b mutant alleles in the FEY251 [pAS25-RPL16B] strain. Whole cell extracts were prepared from cells grown in SGal-Leu (Gal) or shifted to SD-Leu (Glc) for 24 h. Equivalent amounts of cell extracts were separated by SDSPAGE and levels of the L16 r-protein variants (indicated by asterisks) were determined by western blot analysis using anti-L16 antibodies. expression of only one of the two r-protein paralogues normally rescues the growth and ribosome biogenesis defects of the strain lacking both paralogues (discussed in [205]). Thus, to check whether multiple copies of rpl16b-N180 suppressed the absence of L16, we constructed a strain containing a single rpl16b-N180 allele at its natural chromosomal locus. 58 As shown in Fig. 8A, this strain grew as well as a wild-type strain when present in an RPL16A background and similarly as an rpl16a null mutant when present in an rpl16a∆ background. Western blot analysis showed that the truncated L16B-N168 protein was fairly well expressed (Fig. 8B). Moreover, polysome profile analysis showed that the rpl16a∆ rpl16b-N180 strain displayed a mild reduction in the amount of free 60S r-subunits as well as an accumulation of half-mer polysomes (Fig. 8C). This profile was identical to that obtained for an isogenic rpl16a∆ single mutant (data not shown), indicating that when expressed from a plasmid, the rpl16b-N180 construct was in fact suppressing the defects of both rpl16a and rpl16b null alleles. Thus, we conclude that, at least, the last 18 amino acids of yeast L16 protein are fully dispensable for growth and ribosome production under laboratory conditions. Importance of the C-terminal extensión of L16 in 60S r-subunit biogénesis We next wanted to examine in more detail the consequences of assembly of the truncated L16 proteins. First, we studied whether the truncated mutants lead to defects in pre-rRNA processing. To do so, the GAL::HA-RPL16 strain harbouring an empty YCplac111 vector, or YCplac111-borne copy of either wild-type or the truncated versions of L16B were grown in selective SGal medium, and shifted to selective SD medium for different times. RNA was extracted at the different time points and the levels of pre-rRNA intermediates assayed by northern blotting. As shown in Fig. 9, and in agreement with the absence of apparent defects in growth and ribosome production, expression of L16B-N180 led to wild-type levels of pre-rRNAs and mature rRNAs. Therefore, the last 18 amino acids of L16B are also dispensable for pre-rRNA processing under laboratory conditions. However, expression of L16B-N168 and L16B-N147 resulted in pre-rRNA processing defects similar to those found in the L16-depleted strain, including low levels of 27SB and 7S pre-rRNAs and mature 25S rRNAs. Strikingly, the levels of these pre-rRNAs were reduced to a lesser extent in the truncation mutants than in the L16-depleted strain (Fig. 9). Residual levels of full-length L16 in the truncated mutants might have not accounted for the slight increase in these pre-rRNAs since a similar increase did not occur in the GAL-HA-RPL16 strain transformed with a empty vector, which also presented substantial levels of full-length L16 even 24 h after the shift to selective SD medium (see Fig. 7C). 59 Fig. 8. The genomically integrated rpl16b-N180 allele confers a wild-type phenotype. (A) Growth analysis of the indicated strains compared to their isogenic wild-type control. Cells of strains FEY206 (rpl16a∆), FEY209 (rpl16b∆), JFY01 (rpl16b-N180), JFY02 (rpl16a∆ rpl16b-N180) and W303-1A (Wild type) were serially diluted 5- fold and spotted on YPD plates, which were incubated at 30 ºC for 3 days. (B) The indicated strains were grown in liquid YPD medium and harvested at an OD600 of 0.8, whole cell extracts were prepared and equivalent amounts of the cell extracts subjected to western blot analysis with antibodies against the r-proteins L16 and L1. (C) Polysome profile of the double rpl16a∆ rpl16b-N180 mutant. Ten A260 of the whole cell extract from the JFY02 strain were resolved in a 7-50% sucrose gradient. The A254 was continuously measured. Sedimentation is from left to right. The peaks of free 40S and 60S r-subunits, 80S free couples/monosomes and polysomes are indicated. Half-mers are labelled by arrows. 60 Fig. 9. Role of the C-terminal extension of L16 in pre-rRNA processing. The strain FEY251 [pAS25-RPL16B] harbouring the empty YCplac111 plasmid (Vector), or YCplac111-borne wild-type RPL16B or the indicated truncated rpl16b alleles were grown in liquid SGal-Leu and shifted to liquid SD-Leu for the indicated times. Cells were harvested and total RNA extracted. Equal amount (5 µg) of total RNA was separated on an agarose gel (first three upper panels) or on a polyacrylamide gel (lower panels), transferred onto nylon membranes, and hybridized with the indicated probes (between parentheses; probes are described in the Experimental Procedures section). Together, these results strongly suggest that assembly of the non-viable truncated versions of L16B minimally but significantly stabilize early and medium pre-60S r-particles, hence, decreasing their rate of turnover. To determine whether assembly of the truncated L16B variant proteins, similarly as L16 depletion does, impairs nuclear export of pre-60S r-particles, we analyzed the location of the 60S reporter L25-eGFP in the different strains expressing the truncated L16B proteins used in this study. In selective SGal medium, L25-eGFP was found predominantly in the cytoplasm. However, following a shift to selective SD medium for 6 h, L25-eGFP accumulated in the nucle(ol)us in about 90 % of the ca. 200 examined cells expressing L16B-N168 or L16B- N147, this percentage is similar to that obtained upon depletion of L16. In contrast, no nuclear accumulation of L25-eGFP was observed in cells expressing either full-length or L16B-N180 protein (data not shown). We conclude that assembly of the truncated L16B- 61 Fig. 10. Analysis of the incorporation of C-terminally truncated L16 variant proteins into pre-60S/60S rsubunits and translating ribosomes. The strain FEY251 [pAS25-RPL16B] harbouring YCplac111-borne wildtype RPL16B or the indicated truncated rpl16b alleles were grown in liquid SGal-Leu and shifted to liquid SD-Leu for 24 h. Cell extracts were prepared under polysome-preserving conditions and ten A260 units of each extract were resolved in 7–50% sucrose gradients and fractionated. Proteins were concentrated by TCA precipitation and subjected to western analysis using anti-L16 and anti-L5 antibodies. The position of free 40S and 60S r-subunits, 80S free couples/monosomes and polysomes have been obtained from the recorded A254 profile. The corresponding inputs of the fractionated cell extract samples are shown in Fig. 7C. Note that a substantial residual full-length HA-L16 protein is still detected in cells expressing the L16B-N168 and L16B-N147 variants (indicated by asterisks). N168 or L16B-N147 does not ameliorate the impairment of nuclear export of pre-60S rparticles caused by the depletion of L16, while assembly of L16B-N180 fully complements this failure. Finally, to directly examine whether the truncated L16B variant proteins get incorporated into mature ribosomes, we obtained extracts from cells expressing wild-type or the truncated L16B proteins, subjected them to sucrose gradient fractionation, and the fractions analysed by SDS-PAGE and western blotting using polyclonal anti-L16 antibodies. 62 As shown in Fig. 10 and as expected, the full-length L16B and L16B-N180 proteins got efficiently incorporated into mature 60S r-subunits and engaged in active translation. Moreover, both L16B-N168 and L16B-N147 proteins, which inefficiently accumulated as previously shown (see Fig. 7C), were found associated with high-molecular-mass complexes sedimenting at positions equivalent to 80S monosomes and light polysomes (Fig. 10). We conclude that the truncated L16B-N168 or L16B-N147 could also get incorporated into pre- 60S/60S r-subunits, which are likely not as translationally competent as those derived from wild-type or rpl16-N180 cells. 63 EXPERIMENTAL PROCEDURES Strains and microbiological methods The yeast strains used in this study (Table 1) are derivatives of the diploid strain W303 [206]. Heterozygous diploid strains disrupted for one RPL16A ORF copy or one RPL16B ORF copy, respectively, with the HIS3MX6 marker were obtained by following a standard procedure [207]. FEY206 (rpl16a::HIS3MX6) and FEY206 (rpl16b::HIS3MX6) are meiotic segregants the corresponding diploid strains. To construct the GAL::RPL16 strain, FEY206 and FEY206 were crossed and the resulting diploid transformed with a YCplac33- RPL16B plasmid. After sporulation and tetrad dissection, we selected the FEY251 strain, which is a representative double rpl16a∆ rpl16b∆ mutant that contains YCplac33-RPL16B. FEY251 was transformed with the pGAL1-RPL16B plasmid [175], and subsequently, YCplac33-RPL16B was counter-selected on 5-FOA plates containing galactose. We also refer to this strain as GAL::RPL16. C-terminal truncations of RPL16B were performed in the genomic RPL16B locus by a standard replacement procedure [207]. Details about the construction of the different strains are available upon request. Strains were grown at selected temperatures either in rich YP medium (1% yeast extract, 2% peptone) supplemented with 0.2% adenine and containing either 2% glucose (YPD) or 2% galactose (YPGal) as carbon source or in 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 either 2% glucose (SD) or 2% galactose (SGal) as carbon source. Yeast was transformed by the lithium acetate method [208]. Tetrad dissections were performed using a Singer MS micromanipulator. All recombinant DNA techniques were done according to established procedures using E. coli DH5α for cloning and propagation of plasmids. Plasmids To construct YCplac33-RPL16B and YCplac111-RPL16B, a PCR was performed using yeast genomic DNA as a template and oligonucleotides placed plus-minus 1 kbp upstream and downstream from the start-stop codon of the RPL16B ORF, respectively; after the appropriate restriction digestion, the PCR product was cloned into the respective vectors. Different C-terminal truncations of L16B were also cloned into YCplac111. To construct pAS25-RPL16B, which allows expression of N-terminally HA-tagged L16B under the control of a GAL promoter, a PCR product containing the RPL16B ORF plus 500 bp downstream the stop codon was cloned into the empty pAS25 vector [209]. The correctness of all resulting plasmids was confirmed by DNA sequencing. Detailed information on the plasmids is 64 available upon request. Other plasmids used in this study were: pRS316-RPL25-eGFP- NOP1-mRFP and pRS316-RPS3-eGFP-NOP1-mRFP (gifts from J. Bassler and E. Hurt) [210] and pGAL1-RPL16B (TK810) (gift from JP. Milkereit) [175]. Polysome profile analyses Polysome preparations were performed as exactly described in [211]. Sucrose gradients were analysed using an ISCO UA-6 system with continuous monitoring at A254. When required, fractions of 0.5 ml were collected from the gradients and proteins were extracted and analysed by western blotting as described [212]. Western blotting analyses Total cell extracts were obtained by disrupting cells by vigorous shaking with glass beads in a Fastprep-24TM 5G (MP Biomedicals) at 4 ºC, in a standard lysis buffer (50 mM Tris-HCl, pH7.5; 1.5 mM MgCl2; 150 mM NaCl; 1 mM DTT) containing a protease inhibitor cocktail (Complete, Roche); extracts were clarified by centrifugation in a microcentrifuge at the maximum speed (ca. 16100 x g) for 15 min at 4 ºC. Total cell extracts were analysed by western blotting according to a standard procedure using the following primary antibodies: mouse monoclonal anti-Nop1 (MCA28F2, EnCor Biotechnology) and rabbit polyclonal anti- L16 (gift from S. Rospert) [213], anti-L1 (gift from F. Lacroute) [214], anti-Has1 (gift from P. Linder) [215], anti-Mrt4 (gift from J.P.G. Ballesta) [115], anti-L5 (gift from S.R. Valentini) [216] and anti-L10 (gift from B. Trumpower) [217]. Goat anti-mouse or anti-rabbit horseradish peroxidase-conjugated antibodies (Bio-Rad) were used as secondary antibodies. Proteins were detected using a chemiluminescence detection kit (Super-Signal West Pico, Pierce) and a ChemiDocTM MP system (Bio-Rad). RNA extractions, northern hybridization and primer extension analyses RNA extraction, northern hybridization and primer extension analyses were carried out as previously described [218, 219]. Total RNA was extracted from samples corresponding to 10 OD600 units of cells grown to mid-log phase. Equal amounts of total RNA were loaded on gels or used for primer extension reactions. Specific oligonucleotides, were 5'-end labelled with [γ-32P] ATP and used as probes. The following probes were used: Probe a (5' A0) 5'-GGTCTCTCTGCTGCCGG-3', Probe b (18S) 5'-CATGGCTTAATCTTTGAGAC-3', 65 Probe c (3-D/A2) 5'-GACTCTCCATCTCTTGTCTTCTTG-3', Probe d (A2/A3) 5'- TGTTACCTCTGGGCCC-3', Probe e (5.8S) 5'-TTTCGCTGCGTTCTTCATC-3', Probe f (E/C2) 5'-GGCCAGCAATTTCAAGTTA-3', Probe g (C1/C2) 5'-GAACATTGTTCGCCTAGA-3', Probe h (25S) 5'-CTCCGCTTATTGATATGC-3' and Probe 5S 5'- GGTCACCCACTACACTACTCGG-3'. Signal intensities were quantified using a FLA-5100 imaging system and Image Gauge (Fujifilm). Pulse-chase labelling of pre-rRNA Pulse-chase labelling of pre-rRNA was performed exactly as previously described [219], using 100 µCi of [5,6-3H]-uracil (Perkin Elmer; 45 to 50 Ci/mmol) per 40 OD600 of yeast cells. Cells were grown in SGal-Ura medium to mid-log phase at 30 ºC or shifted to SD-Ura medium for 8 h, pulse-labeled for 2 min and chased for different times with SD medium containing an excess of cold uracil. Total RNA was extracted as above. Radioactive incorporation was measured by scintillation counting and about 20000 cpm per RNA sample were loaded and resolved on 1.2% agarose-6% formaldehyde and 7% polyacrylamide-8M urea gels. RNA was then transferred to nylon membranes and visualized by fluorography [219]. Fluorescence Microscopy To test pre-ribosomal particle export, the appropriate GAL::RPL16 strain derivatives were transformed with plasmids expressing either GFP-tagged L25 or S3 and Nop1-mRFP (see above). Transformants were grown in selective SGal medium and shifted to selective SD for 6 h to deplete L16. Cells were washed, resuspended in PBS buffer (140 mM NaCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 2.75 mM KCl, pH 7.3), and examined with a Leica DMR fluorescence microscope equipped with a DC350F digital camera and its software. Adobe Photoshop CS2 (Adobe Systems Inc.) was used to process the images. Affinity Purification of GFP-tagged Proteins GFP-tagged factors representative of 90S and pre-60S r-particles were purified following a one-step GFP-Trap_A procedure (Chromotek), as exactly described in [134]. The proteins from purified particles were extracted by boiling the beads with Laemmli buffer and analyzed by western blotting. To normalize the amount of purified complexes to be loaded for comparative studies, aliquots of the samples were first resolved and visualized by silver staining according to the manufacturer’s instructions (Bio-Rad). 72 Fig 1. Pre-rRNA processing pathway in S. cerevisiae. (A) Structure and processing sites of the 35S and pre- 5S pre-rRNAs. Mature 18S, 5.8S and 25S rRNAs are flanked by external transcribed (ETS) and separated by internal transcribed (ITS) spacer sequences; pre-5S rRNA is separated from 35S pre-rRNA by non-transcribed sequences (NTS). Processing sites and location of the different probes used in this study are indicated. (B) PrerRNA processing pathway. Endo- and exonucleolytic reactions are indicated. The known nucleases responsible of the processing reactions are highlighted. For more details on yeast pre-rRNA processing, see [35, 48, 181]. A A B 73 eL29, eL40, eL42, P1 and P2, respectively), which are located at the r-subunit interface (for references, see [78, 125, 140, 257]). As for 40S r-subunits, the sequence of r-protein incorporation into 60S r-subunits has been well conserved in higher eukaryotes [258]. The role of few r-proteins on pre-rRNA processing and ribosome assembly remains undefined or has only been poorly characterised. Among these orphan proteins is L14 (eL14). L14 is an eukaryote-specific r-protein from the 60S r-subunit, which is conserved in some but not all archaea [222, 259]. In addition, eukaryotic L14 contains a specific C- terminal extension, absent in the archaeal orthologues (see [176]), whose role is unknown. L14 forms a eukaryote-specific structure, close to the r-stalk, on the solvent-exposed surface of the 60S r-subunits together with the ES7L and ES39L of domain II and VI of 25S rRNA, respectively, H41 of domain II of 25S rRNA, its interacting r-proteins L6, L9, L16, L20, and its neighbouring r-proteins L7, L32 and L33. (Figure 2). Knockdown of RPL14 expression by Fig. 2. Molecular environment of L14 in the yeast 60S r-subunit. Position of L14 and its rRNA/protein neighbourhood in the solvent-exposed surface of the yeast 60S r-subunit. The r-proteins L6 (salmon), L7 (cyan), L9 (green), L14 (red), L16 (marine blue), L20 (light blue), L32 (brown) and L33 (middle blue) are shown. The rRNA expansion segments ES7L and ES39L are shown in gold and 5.8S rRNA in red. The remaining 60S rproteins are coloured in black and rRNA in pale grey. The representation were rendered with UCSF Chimera [193], using the atomic model for the crystal structure of the yeast 80S ribosome at 3.0 Å (PDB ID: 3U5I and 3U5H; [10]). Note that ES7La is not resolved in this model. 74 siRNA in human HeLa or HCT116 cancer cells impairs 28S and 5.8S rRNA synthesis and formation of 60S r-subunits [139, 258]. In this study, we have examined the precise role of the otherwise uncharacterised yeast L14 r-protein in ribosome biogenesis. Our data indicate that yeast L14 assembles in the nucleolus within earliest pre-60S r-intermediates. Depletion of L14 causes a shortage of 60S r-subunits, which is due to defective production and accelerated turnover of early and intermediate pre-60S r-particles. Consistently, pulse-chase analyses demonstrate impaired production of mature 25S and 5.8S rRNAs, northern blot hybridization shows mild accumulation of 27SA pre-rRNAs and low steady-state of 27SB and 7S pre-rRNAs, and fluorescence microscopy suggests a defect in the nuclear export of pre- 60S r-particles. We have also analysed the impact of the L14 depletion on the protein composition of early pre-60S r-particles, which were affinity-purified using TAP-tagged Noc2 as a bait. Our results clearly show that L14 is required for the stably assembly of a subset of r-proteins, mainly located in its immediate neighbourhood, such as L6, L20 and L33 or surrounding the solvent-exposed part of the peptide exit tunnel such as L17, L26, L37 and L39. L14 is also particularly required for the association of few late-acting ribosome assembly factors, most of them essential for cleavage at site C2 in ITS2. Finally, we also discuss on the role of the eukaryote-specific C-terminal extension of yeast L14 in ribosome biogenesis and function. 75 RESULTS Yeast L14 assembles in the nucle(ol)us within early pre-60S r-particles Assembly of most r-proteins occurs primarily in the nucle(ol)us, although few ones appear to load preferentially or exclusively in the cytoplasm (reviewed in [78]). No specific information was available on the course of the incorporation of yeast L14 into pre-60S rparticles. We suspect it should occur into the nucleus despite the fact that no nuclear localisation sequence can apparently be predicted in L14 (cNLS mapper programme, [260]; NucPred tool, [261]). Indeed, one of us has previously shown that L14 is included in the broad list of r-proteins that are roughly equally enriched in early to late pre-60S r-particles [125]. Consistently, L14 has been identified at its final location in cryo-EM maps of distinct nuclear pre-60S r-particles (e.g. [153, 262, 263]). Fig 3. Functional analysis of the L14A-yGFP construct. (A) Strains W303-1B (Wild type) and FEY229, which is a rpl14A∆ rpl14B∆ null strain, expressing either L14A (RPL14A) or L14A-yGFP (RPL14A-yGFP) from a centromeric YCplac111 plasmid were grown in YPD and diluted to an OD600 of 0.05. A 5-fold series of dilutions were performed and 5 l drops were spotted onto YPD plates. Plates were incubated at 30 ºC for 3 days. (B) Polysome profiles of the RPL14A and RPL14A-yGFP strains. These strains were grown in YPD at 30 °C to an OD600 of 0.8. Cell extracts were prepared, and 10 A260 of each extract were resolved in 7-50% sucrose gradients. The A254 was continuously measured. Sedimentation is from left to right. The peaks of free 40S and 60S rsubunits, 80S vacant ribosomes/monosomes, and polysomes are indicated. A A B 76 To explore in further detail the timing of incorporation of L14 into pre-60S r-particles, we first monitored the localisation of an L14A-yGFP construct upon induction of dominant negative NMD3-∆100 allele, which has been proven to trap nascent pre-60S r-particles in the nucleus [264]. The L14A-yGFP construct fully complemented the growth and ribosome biogenesis defects of the null rpl16A∆ rpl16B∆ strain (Figure 3). As shown in Figure 4, L14A-yGFP accumulated in the nucleus of most of the cells examined upon overexpression of the Nmd3-∆100 protein but it was found in the cytoplasm under non-inducible conditions. No changes in the cytoplasmic distribution of L14A-yGFP was observed upon overexpression of a wild-type Nmd3 protein (data not shown). Fig. 4. Nuclear assembly of L14A-yGFP. Localization of L14A-yGFP upon induction of the dominantnegative NMD3∆100 allele. Cells of the rpl14 null strain expressing GFP-tagged L14A as the sole source of L14 was transformed with the pRS316-GAL-NMD3∆100 plasmid. Transformants were grown in SD-Ura (Glc) or shifted for 12 h to SGal-Ura (Gal) to induce the Nmd3∆100 protein. The GFP signal was inspected by fluorescence microscopy. Arrows point to nuclei. Approximately 200 cells were examined for the reporter and practically all cells gave the results shown in the representative pictures. Next, we tested for the presence of L14 within different GFP-affinity purified preribosomal particles on the road of maturation of 60S r-subunits. The specificity of the purification was evaluated by assessing the presence of the selected trans-acting factors Nop1, Has1 and Mrt4 by western blot using specific antibodies (Figure 5). Western blot analyses also showed that L14 significantly co-enriched with practically all tested particles (Figure 5), as did L1, which was used as a control of an early assembling r-protein [125, 166]. In contrast, L10, which has been reported to assemble in the cytoplasm [125, 200], only co-enriched with late and cytoplasmic pre-60S r-particles and P0-GFP-containing particles that correspond mainly to mature ribosomes [134]. Interestingly, L14 seemed not to be as 77 stably associated with early 90S particles, which were purified using GFP-tagged Pwp2/Utp1 as L16 or L1; similar results to those of L14 were obtained for L6, a r-protein that directly interacts with L14 [265], which was also found even poorer represented in 90S/66S particles purified using GFP-tagged Nop58/Nop5 than L14 (Figure 5). Together, these data indicate that L14 assembles in early nucle(ol)ar pre-60S r-particles. Fig. 5. Association of L14 with maturing 60S r-subunits. 90S and pre-60S r-particles from early to late intermediates were affinity-purified by using the GFP-Trap immunoprecipitation procedure with the indicated bait proteins. Equivalent amounts of immunoprecipitates were analyzed by 12% SDS-PAGE and western blot analysis. Specific antibodies were used to detect the Nop1, Has1 and Mrt4 trans-acting factors and the L1, L6, L10, L14 and L16 r-proteins. Note that the Rix1-GFP and Lsg1-GFP are slightly underloaded. The affinity-purified complexes and all protein signals, except those of L14 and L6, have been previously shown in [137]. In agreement with these results, when we affinity purified L14A-yGFP-containing particles and assayed those pre-rRNA intermediates that co-purified by northern blot hybridization, we detected 27SB and 7S pre-rRNAs clearly above background levels (Figure 6). As previously shown for other 60S r-proteins [131, 133, 136], L14A-yGFP also co-purified substantial amounts of mature rRNAs (Figure 6). The 27SA2 pre-rRNA was also detected, albeit slightly above background levels (Figure 6), suggesting that either L14A associates with pre-60S r-particles once cleavage at site A2 has occurred or more likely that the assembly of L14A is only stabilised after some reorganization of the pre-rRNAs that takes place concomitantly to the formation of 27SB pre-rRNAs. Taken together, all these results suggest that L14 assembles into early pre-60S r-particles and appears to become stable only in those containing the 27SB pre-rRNAs. 78 Fig. 6. L14-yGFP stably assembles within pre-60S r-particles containing 27SB pre-rRNAs. Affinitypurification of ribosomal particles was performed from cells expressing L14A-yGFP via the GFP-Trap immunoprecipitation procedure. Wild-type cells (untagged L14) were used as a negative control. Total RNA was extracted from whole cell extracts (T) and immunoprecipitates (IP) and analysed by northern blotting. (A) Northern blot analysis of high-molecular-mass or (B) low-molecular-mass pre- and mature rRNAs. The indicated probes, between parentheses, were used to detect the different pre- and mature rRNA species (see Fig. 1A and Table 3). L14 is required for normal accumulation of 60S ribosomal subunits L14, as most r-proteins (e.g. [266]), is essential for cell viability, and is encoded by two different paralogous genes, RPL14A and RPL14B. Deletion of RPL14A caused a severe defect on growth, while deletion of RPL14B led to a minor growth defect (Figure 7A, see also [266]). These defects seemed to be exacerbated at lower temperatures (Figure 7A). As shown in Figure 7B, both deletions led to polysome profiles alike to those for mutants showing a deficit on the content of 60S r-subunits. Thus, for the profiles of both deletion strains, we observed a decrease in the amount of free 60S r-subunits relative to free 40S rsubunits and the appearance of half-mer polysomes. The overall defects were apparently more pronounced for the rpl14A∆ than for the rpl14B∆ null strain. These data are in agreement with those previously described by [176]. A A B 79 Fig 7. Deletion of either RPL14A or RPL14B leads to a deficit in 60S r-subunits. (A) Growth phenotypes of the rpl14A and rpl14B null mutants. Strains W303-1B (Wild type), FEY204 (rpl14AΔ), FEY203 (rpl14BΔ) were grown in YPD and diluted to an OD600 of 0.05. A 5-fold series of dilutions were performed and 5 l drops were spotted onto YPD plates. Plates were incubated at 22 ºC, 30 ºC and 37 ºC for 3 days. (B) Polysome profiles of the above strains. These strains were grown in YPD at 30 ºC to an OD600 of 0.8. Cell extracts were prepared, and 10 A260 of each extract were resolved in 7-50% sucrose gradients. The A254 was continuously measured. Sedimentation is from left to right. The peaks of free 40S and 60S r-subunits, 80S vacant ribosomes/monosomes, and polysomes are indicated. Half-mers are labeled by arrows. For the phenotypic analysis of the complete loss-of-function of L14, we constructed a conditional strain, named GAL::RPL14 strain, that expressed a double HA-tagged variant of L14A as the sole source of L14 under the control of a GAL promoter. This strain grew as the B A A 80 isogenic wild-type strain or a isogenic GAL::RPL16 control strain (see [137]) on YPGal plates but was unable to grow on YPD plates (Figure 8A). After shifting a culture of the Fig. 8. Depletion of L14 results in a deficit in 60S r-subunits. (A) Growth comparison of the strains W303-1B (Wild type), FEY251 [pAS24-RPL16B] (GAL::RPL16) and FEY229 [pAS24-RPL14A] (GAL::RPL14). The cells were grown in YPGal and diluted to an OD600 of 0.05. A 5-fold series of dilutions were performed and 5 l drops were spotted onto YPGal (Gal) and YPD (Glc) plates. Plates were incubated at 30 ºC for 3 days. Note that the growth of wild-type and GAL::RPL16 strains has been previously shown in [137]. (B) Polysomes profile analysis of GAL::RPL14 cells grown in YPGal (Gal) or shifted to YPD (Glc) for 3 h. Cells were harvested at an OD600 of around 0.8, whole cell extracts were prepared and 10 A260 units of each extract were resolved in 7-50% sucrose gradients. The A254 was continuously measured. Sedimentation is from left to right. The peaks of free 40S and 60S r-subunits, 80S vacant ribosomes or monosomes and polysomes are indicated. Half-mers are labelled by arrows. GAL::RPL14 strain from liquid YPGal to YPD medium, the rate of cell division slowed down significantly in few hours and practically stopped after 8-10 h in YPD (data not shown). When we analysed polysome profiles from cell extracts of the GAL::RPL14 strain grown in A A B 81 YPGal, normal wild-type profiles were obtained (Figure 8B). However, when shifted for about 3 h to YPD, extracts showed abnormal polysome profiles, consisting of a clear decrease in the levels of free 60S versus free 40S r-subunits, a decrease in the 80S peak and polysomes and the appareance of half-mer polysomes (Figure 8B). Taken together, these results indicate that L14 is essential for growth and for normal accumulation of 60S rsubunits. L14 is required for 27S pre-rRNA processing and normal production of 25S and 5.8S rRNAs To study whether the deficit of 60S r-subunits caused by the depletion of L14 is attributed to a defective production or/and an excessive degradation of 60S r-subunits, we analysed the effects of L14 depletion on the synthesis and processing of pre-rRNAs by [3H]uracil pulse-chase labelling experiments. For this purpose, the GAL::RPL14 and an isogenic wild-type control strain were grown in YPGal and shifted to SD-Ura for 6 h. In wildtype cells, the 35S pre-rRNA was processed rapidly into 32S pre-rRNA, and then into 27S and 20S pre-rRNAs, which were subsequently converted into mature 25S and 5.8S, and 18S rRNAs, respectively (Figure 9). In contrast, in the GAL::RPL14 strain, processing of the 35S pre-rRNA was delayed; as a consequence, less 27SA and 20S pre-rRNAs were formed and traces of 23S pre-rRNA were detected, but while enough mature 18S rRNA was still made, some 27SB pre-rRNAs persisted even after the 60 min of chase and practically no labelled mature 25S and 5.8S rRNAs were detected (Figure 9). These defects were apparently specific since no changes in the kinetics of production of mature 5S rRNA or tRNAs or their levels were observed upon the depletion of L14 (Figure 9). These results indicate that the deficit in 60S r-subunits following depletion of L14 was due to impaired processing and increased turnover of 27SB pre-rRNAs. As a consequence, reduced levels of both mature 25S and 5.8S rRNAs were synthesized. Most likely as a side effect (for further discussion, see [137] and references therein), earlier processing reactions at sites A0, A1 and A2 were also delayed upon depletion of L14; however, this delay impacted only slightly 18S rRNA production. 88 Fig 13. Ribosomal proteins that significantly diminish from Noc2-TAP containing pre-ribosomal particles upon depletion of L14. Different subset of r-proteins are shown: (i) L14 (red), (ii) L6, L16, L20, L33 (different blue tones), which are r-proteins nearby L14 that fail to associate upon depletion of L14. (iii) L17, L19, L26, L37 and L39 (different green tones), which are r-protein that surround the polypeptide exit tunnel. (iv) L28, L36, L34 and L2 (different pink tones). 5.8S rRNA was coloured in red and 5S in gold. Note that a structure for any early pre-60S rparticles has still not been determined. Thus, r-proteins were represented using the atomic model for the crystal structure of the yeast 80S ribosome at 3.0 Å (PDB ID: 3U5I and 3U5H; [10]). L14 is required for the stable association of a set of 60S r-subunit biogenesis factors required for processing of 27SB pre-rRNA We also determine how the depletion of L14 affected the association of trans-acting factors to Noc2-TAP affinity-purified pre-ribosomal particles. As shown in Figure 12B, very few factors diminished to a different extent. Most of these factors (also known as B-factors), Spb4, Dbp10, Spb1, Nsa2, Tif6, Nip7, Nop2 and Rlp24, are required for 27SB pre-rRNA processing, thus for cleavage at site C2 within ITS2 [146, 160, 212, 272-277]. Dbp10, Tif6, Rlp24 are among the subset of factors required to recruit Nsa2 to pre-60S r-particles [272]. Nip7 and Nop2 form a heterodimer [272]. Nug1, which is apparently not a B-factor, genetically interacts with and is required for the stable association of Dbp10 with preribosomal particles [278, 279]. 89 Few other factors required for 27SB pre-rRNA processing are enriched upon depletion of L14, among them, Nog1 and the physical and functional related pair of factors Rpf2 and Rrs1 [130], which guides assembly of the 5S rRNP (see [255], and references therein). The significance of the enrichment of Rrp14, Nop4, Nop16, Rsa3, Puf6 and Noc3 is still unclear, although it has been described that Rrp14 delays 27SB pre-rRNA processing and interacts with Epb2 in a two-hybrid assay [280]. Remarkably, practically all A3-factors so far described were found among the proteins that enriched upon depletion of L14: Has1, Drs1, Rrp1, Nop12, Brx2, Epb2, Nop7, Ytm1, Erb1, Nsa3/Cic1, Nop15 and Rlp7 (for details, see [30]). Depletion of any of these factors leads to defect in 27SA3 pre-rRNA processing (e.g. [132]), phenotype that it is the most significant feature of the L14 depletion (see Figure 10C). It has been shown that factors Nop7, Ytm1 and Erb1 form a stable sub-complex to which Drs1 associates [281, 282]. This sub-complex together with Nsa3, Nop15 and Rlp7 has been described to be mutually interdependent for association with pre-60S r-particles [132]. Has1 interacts with Nop15 in a two-hybrid assay [283]. In conclusion, depletion of L14 prevents stable association of a set of factors required for 27SB pre-rRNA processing with early pre-60S r-particles. Consequently, these particles are unable to progress in their maturation at ITS2 of the pre-rRNAs. In contrast, these particles still associate with most factors required for 27SA3 pre-rRNA processing, but strikingly, they are also unable to support this pre-rRNA processing reaction, as evidenced for the pre-rRNA processing defects observed for a L14-depleted strain. Likely as the direct consequence of these impairments, early pre-60S r-particles are subjected to nuclear retention and efficient turnover following depletion of L14. Disruption of the interaction between the eukaryote-specific C-terminal extensions of L14 and L16 r-proteins causes paromomycin hypersensitivity In both late pre-60S r-particles and mature 60S r-subunits, an interaction between the most distal -helices of the eukaryote-specific C-terminal extensions of L14 and L16 is produced. This interaction is maintained by apolar contacts between amino acids of both helices and two salt bridges, one between the carboxylic group of L14[A138] and the -amino group of L16[K177] and another between the guanidinium group of L14[R109] and the carboxylic group of L16[Y199] (Figure 2). To study the relevance of this interaction in cell growth and ribosome biogenesis, we tested the genetic interaction between two distinct and previously reported truncated alleles: rpl14A-L123*, which expressed a truncated L14A variant lacking the last 16 amino acids [176] and rpl16B-N180, which expressed a truncated L16B variant lacking the last 18 amino acids [137]. These two alleles confer a practically 90 wild-type phenotype at 30 ºC but a 60S r-subunit deficit when expressed as the sole copy of each respective r-protein in yeast cells [137, 176] (see also Figure 14A). Moreover, combination of these two alleles as the sole sources of L14 and L16 in double mutant cells did not cause any synergistic growth defect phenotype in either rich or synthetic media at either 30 ºC, 37 ºC or 22 ºC (Figure 14A and data not shown), and both protein variants were expressed to wild-type levels (Figure 14B). Interestingly, the double mutant but neither of the single ones displayed increased sensitivity to paramomycin (Figure 14A). This observation suggest that, despite, its practically wild-type growth rate at 30 ºC, cells lacking the most distal parts of the C-terminal extensions of L14 and L16 produce 60S r-subunits that might be enough structurally abnormal to manifest slight dysfunctions when subjected to certain stresses, such as the presence of distinct antibiotics that inhibit translation. Fig. 14. The rpl14A-N123 rpl16B-N180 mutant is hypersensitive to paromomycin. (A) Growth phenotypes of the indicated strains on YPD plates without antibiotics or containing 5 mg/ml neomycin or 1 mg/ml paromomycin. A triple rpl14A∆ rpl14B∆ rpl16A∆ null mutant containing the indicated combinations of plasmid-borne RPL14A and rpl14A-N123 allele and genomic RPL16B and rpl16B-N180 alleles as sole sources of L14 and L16 respectively, were grown in YPD medium and spotted in 10-fold serial dilution steps onto YPD plates with or without the indicated antibiotics. Plates were incubated at 30 ºC for 3 days. (B) Expression levels of the plasmid-borne wildtype and indicated mutant alleles of both RPL14A and RPL16B genes as sole sources of L14 and L16. Equivalent amounts of cell extracts of the above strains were separated by SDS-PAGE and levels of the L14 and L16 rproteins variants were determined by western blot analysis using specific anti-L14 and anti-L16 antibodies. A A B 91 EXPERIMENTAL PROCEDURES Strains and microbiological methods The S. cerevisiae strains used are listed in Table 1 and are derived from W303 [206]. The deletion disruption of the RPL14A and RPL14B genes and the C-terminal TAP-tagging of NOC2 at the genomic locus were performed as previously described [125, 207, 284]. FEY204 (rpl14A::HIS3MX6) and FEY203 (rpl14B::HIS3MX6) are meiotic segregants of the corresponding diploid strains. To construct the GAL::RPL14 strain, FEY203 and FEY204 were crossed and the resulting diploid transformed with the YCplac33-RPL14A plasmid. After sporulation and tetrad dissection, we selected the FEY229 strain, which is a representative double rpl14A∆ rpl14B∆ mutant that contains YCplac33-RPL14A. Then, FEY229 was transformed with the pAS24-RPL14A plasmid, and subsequently, YCplac33-RPL14A was counter-selected on 5-FOA plates containing galactose. The correct genomic integration of the TAP-URA3 cassette in the FEY320 strain was verified by PCR and the expression of the Noc2-TAP fusion protein by western blot analysis. Strains JFY136 and JFY137 are also meiotic segregants; YKL516 and JFY01 were crossed, the resulting diploid was sporulated, and tetrads were dissected. In spore clones with the appropriate markers, the presence of the wild-type or mutant variant alleles of RPL14A/B and RPL16A/B genes were analysed by PCR. The expression of the wild-type L14A and L16B or the truncated variants L14A-N123 and L16B-N180 proteins was tested by western blot analysis. Strains were grown at selected temperatures either in rich YP medium (1% yeast extract, 2% peptone) supplemented with 0.2% adenine and containing either 2% glucose (YPD) or 2% galactose (YPGal) as carbon source or in 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 either 2% glucose (SD) or 2% galactose (SGal) as carbon source. Preparation of media and genetic manipulations were done according to established procedures. Antibiotic-containing plates were prepared by adding the drugs from stocks solution into 2% YPD-agar medium before pouring the plates [133]. Yeast cells were transformed by the lithium acetate method [208]. Tetrad dissections were performed using a Singer MS micromanipulator. Plasmids All recombinant DNA techniques were done according to established procedures using E. coli DH5 for cloning and propagation of plasmids. Plasmids used in this study are listed in Table 2. To construct YCplac33-RPL14A, YCplac111-RPL14A, pAS24-RPL14A and 92 YCplac111-RPL14A-yGFP, specific DNA fragments were PCR-amplified using yeast genomic DNA as a template and the appropriate oligonucleotides. The oligonucleotides used in this study are listed in Table 3. After restriction digestion, the PCR products were cloned into the respective vectors. All cloned DNA fragments generated by PCR were verified by sequencing. Detailed information on the plasmids is available upon request. YCplac22- RPL14A and YCplac22-rpl14A-N123, a generous gift from D. Kressler, have been previously reported [176]. Other plasmids used in this study were: pRS314-RPL25-eGFP-NOP1-mRFP and pRS314-RPS3-eGFP-NOP1-mRFP, gifts from J. Bassler and E. Hurt, [210] and pRS316-GAL-NMD3∆100, gift from A. Jacobson [285]. Polysome profile analyses Cell extracts for polysome proflle analyses were performed as described previously [211]. 7-50% sucrose gradients were centrifuged at 39,000 rpm in a Beckman Coulter rotor SW41 Ti at 4 ºC for 2 h 45 min and fractionated using a Teledyne-ISCO UA-6 system with continuous monitoring at A254. Pulse-chase labeling of pre-rRNA Pulse-chase labelling of pre-rRNA was performed exactly as previously described [219], using 100 µCi of [5,6-3H] uracil (Perkin Elmer; 45 to 50 Ci/mmol) per 40 OD600 of yeast cells. Cells were grown in SGal-Ura medium at 30 ºC to mid-log phase or shifted to SD-Ura medium for 6 h, pulse-labeled for 2 min and chased for different times with SD medium containing an excess of cold uracil. Total RNA was extracted by the hot acidic phenolchloroform procedure [286]. Radioactive incorporation was measured by scintillation counting and about 20,000 cpm per RNA sample were loaded and resolved on 1.2% agarose-6% formaldehyde and 7% polyacrylamide-8M urea gels. RNA was then transferred to nylon membranes and visualized by fluorography [219]. Steady-state analysis of pre-rRNA Northern hybridization and primer extension analyses were carried out as previously described [218, 219]. Total RNA was extracted from samples corresponding to 10 OD600 units of cells grown to mid-log phase. Equal amounts of total RNA were loaded on gels or used for primer extension reactions. Specific oligonucleotides (see Table 3), were 5'-end labelled with [-32P] ATP and used as probes. Hybridization signals were detected using a Typhoon™ FLA9400 imaging system (GE Healthcare) and quantified using the GelQuant.NET software (biochemlabsolutions.com). 93 Affinity Purification of GFP-tagged Proteins GFP-tagged factors representative of 90S and pre-60S r-particles were purified following a one-step GFP-Trap_A procedure (Chromotek), as described previously [220]. The proteins from purified particles were extracted by boiling the beads with Laemmli buffer and analyzed by western blotting. To normalize the amount of purified complexes to be loaded for comparative studies, aliquots of the samples were first resolved by SDS-PAGE and visualized by silver staining. Affinity purification of particles containing L14A-yGFP was also performed by the onestep GFP-Trap_A procedure. The pre- and mature rRNAs associated with the particles were recovered by the hot phenol-chloroform method and analysed by northern blotting as described above. Fluorescence Microscopy To test pre-ribosomal particle export, the GAL::RPL14 strain was transformed with plasmids expressing either GFP-tagged L25 or S3 and Nop1-mRFP (see above). Transformants were grown in selective SGal medium and shifted to selective SD for 6 h to deplete L14. Cells were washed, resuspended in PBS buffer (140 mM NaCl, 8 mM Na2HPO4, 1.5 mM KH2PO4, 2.75 mM KCl, pH 7.3), and examined with an Olympus BX61 fluorescence microscope equipped with a digital camera. Images were analysed using the Olympus cellSens software and processed with Adobe Photoshop CS2 (Adobe Systems Inc.). Protein extracts and western blotting analyses Total yeast protein extracts were prepared as described previously [137]. Total protein extracts and samples from purified particles were analysed by western blotting according to standard procedures. The following primary antibodies were used in this study: mouse monoclonal anti-GFP (clones 7.1 and 13.1, Roche) and anti-Nop1 (MCA28F2, EnCor Biotechnology); rabbit polyclonal anti-L1 (gift from F. Lacroute) [214], anti-L6 (gift form G, Dieci) [287], anti-L10 (gift from B. Trumpower) [217], anti-L14 (gift from G. Dieci) [287], anti- L16 (gift from S. Rospert) [213], anti-Has1 (gift from P. Linder) [215], anti-Mrt4 (gift from J.P.G. Ballesta) [115]. Secondary goat anti-mouse or anti-rabbit horseradish peroxidaseconjugated antibodies (Bio-Rad Laboratories, Inc.) were used as secondary antibodies. Proteins were visualized using a chemiluminescence detection kit (Super-Signal West Pico, Pierce) and a ChemiDoc™ MP imaging system (Bio-Rad Laboratories, Inc.). Images were processed with Adobe Photoshop CS2 (Adobe Systems Inc.). 94 Affinity-purification of Noc2-TAP containing pre-ribosomal particles The FEY320 strain, which expresses a chromosomally-encoded Noc2-TAP fusion protein and harbours a GAL::HA-RPL14A allele as the sole source of L14 r-protein, was grown in YPGal to an OD660 of ca. 0.8 and shifted to YPD for 6-8 h to shut down RPL14A expression. Noc2-TAP and associated pre-ribosomal particles were then affinity purified from total cell extracts using a matrix of rabbit IgG covalently coupled to magnetic beads (Sigma- Aldrich) as described previously [125] with some minor modifications. The cell pellet corresponding to one litre of yeast culture was resuspended in 1.5 ml of cold MB buffer (20 mM Tris HCl pH 8,0, 200 mM KCl, 5 mM (CH3COO)2Mg, 2 mM benzamidine, 1 mM PMSF, and 0.02 U/ml RNasin®) per g of cell pellet. Cells were broken by vigorous shaking with glass beads (1.4 g beads of 0.75-1 mm diameter per 0.8 ml of cell suspension) in a Vibrax VXR shaker (IKA) for 10 min at 4°C, followed by 2 min on ice. This procedure was repeated twice. Total cell extracts were clarified by two consecutive centrifugation steps in a microfuge at the maximum speed (ca. 16000 x g) for 5 min and 10 min, respectively. The protein concentration of the cleared lysated was determined using the Bradford protein assay (Bio- Rad). To each of the resulting supernatant (typically 1 ml with ca. 50 mg of total protein), Triton X-100 (0.5% final), Tween 20 (0.1% final) and 200 ml of IgG-coupled magnetic beads slurry, equilibrated with MB buffer containing 0.5% Triton X-100 and 0.1% Tween, were added, and the mixture was incubated for 2 h 30 min at 4 ºC with end-over-end tube rotation. After incubation, the beads were washed four times with 700 µl cold MB buffer with 0.5% Triton X-100 and 0.1% Tween 20. RNA was extracted from the 20% of the mixture to perform northern blot analyses. The remaining 80% mixture was washed two times with 1 ml AC buffer (100 mM CH3COONH4, pH 7.4, 0.1 mM MgCl2) to remove excess of salt from the samples and bound proteins eluted two times with 500 µl of freshly prepared 500 mM NH4OH solution for 20 min at room temperature, pooled and lyophilised overnight. Semiquantitative mass spectrometric analyses Lyophilised eluates were further processed for iTRAQ (isobaric tags for relative and absolute quantitation) and mass spectrometric protein analyses, as previously described [288]. Proteins identified by only one peptide were discarded. iTRAQ ratios (depleted versus non-depleted sample) were determined and normalized to the ratio of the Noc2 bait protein. 95 Table 1. Yeast strains used in this study. Strain Relevant genotype Source W303-1A MATa ade2-1 his3-11,15 leu2-3,112 trp1-1 ura3-1 [206] W303-1B As W303-1A but MAT [206] FEY204 As W303-1A but rpl14A::HIS3MX6 This study FEY203 As W303-1B but rpl14B::HIS3MX6 This study FEY229 (a) As W303-1B but rpl14A::HIS3MX6 rpl14B::HIS3MX6 [YCplac33-RPL14A] This study YKL516 (b) As W303-1A but rpl14A::natNT2 rpl14B::HIS3MX6 [YCplac33-RPL14A] [176] FEY320 As FEY229 but noc2::NOC2-TAP-URA3 [pAS24-RPL14A] This study JFY01 As W303-1B but rpl16A::HIS3MX6 rpl16B-N180::HIS3MX6 [137] JFY137 (a) As W303-1B but rpl14A::natNT2 rpl14B::HIS3MX6 rpl16A::HIS3MX6 [YCplac22-RPL14A] This study JFY136 (a) As W303-1B but rpl14A::natNT2 rpl14B::HIS3MX6 rpl16A::HIS3MX6 rpl16B-N180::HIS3MX6 [YCplac22-RPL14A] This study YMD5 (c) As W303-1A but PWP2-GFP(S65T)::kanMX6 [220] YMD24 (c) As W303-1A but NOP58-GFP(S65T)::TRP1 [220] JDY850 As W303-1A but SSF1-GFP(S65T)::natNT2 [220] JDY854 As W303-1A but NSA1-GFP(S65T)::natNT2 [137] JDY851 As W303-1A but NOP7-GFP(S65T)::natNT2 [220] JDY852 As W303-1A but RIX1-GFP(S65T)::natNT2 [220] JDY855 As W303-1A but ARX1-GFP(S65T)::natNT2 [220] JDY853 As W303-1A but LSG1-GFP(S65T)::natNT2 [220] JDY861 (d) As W303-1A but RPP0-GFP(S65T)::natNT2 [220] (a) Depending on the experimental conditions, the corresponding plasmid was replaced by other plasmids containing different alleles of RPL14A. (b) Gift from D. Kressler. (c) Gift from M. Dosil. (d) Gift from M. Remacha. 96 Table 2. Plasmids used in this study. Name Relevant information Source YCplac33 CEN, URA3 [289] YCplac111 CEN, LEU2 [289] YCplac33-RPL14A RPL14A, CEN, URA3 This study YCplac111-RPL14A RPL14A, CEN, LEU2 This study YCplac22-RPL14A (a) RPL14A, CEN, TRP1 [176] YCplac22-rpl14A-N123 (a) rpl14A-N123, CEN, TRP1 [176] pAS24 (b) promoter GAL1-10, N-terminal 2xHA tag, CEN, LEU2 [209] pAS24-RPL14A GAL::2xHA-RPL14A, CEN, LEU2 This study YCplac111-yGFP/TCYC1 C-terminal yGFP tag, TCYC1, CEN, LEU2 D. Kressler YCplac111-RPL14A-yGFP RPL14A-yGFP, TCYC1, CEN, LEU2 This study pFA6a-HIS3MX6 HIS3MX6 cassette [207] pRS314-RPL25-eGFP- NOP1-mRFP (c) RPL25-eGFP, NOP1-mRFP, CEN, TRP1 [210] pRS314-RPS3-eGFP- NOP1-mRFP (c) RPS3-eGFP, NOP1-mRFP, CEN, TRP1 [210] pRS316-GAL-NMD3∆100 (d) GAL::NMD3∆100, CEN, URA3 [285] (a) Gift from D. Kressler. (b) Gift from M. N. Hall. (c) Gift from J. Bassler and E. Hurt. (d) Gift from A. Jacobson. 97 Table 3. Oligonucleotides used in this study. Name 5'-3' Sequence Use Probe a (5' A0) GGTCTCTCTGCTGCCGG pre-rRNA hybridization Probe b (18S) CATGGCTTAATCTTTGAGAC 18S rRNA hybridization Probe c (3-D/A2) GACTCTCCATCTCTTGTCTTCTTG pre-rRNA hybridization Probe d (A2/A3) TGTTACCTCTGGGCCC pre-rRNA hybridization Probe e (5.8S) TTTCGCTGCGTTCTTCATC 5.8S rRNA hybridization Probe f (E/C2) GGCCAGCAATTTCAAGTTA pre-rRNA hybridization Probe g (C1/C2) GAACATTGTTCGCCTAGA pre-rRNA hybridization Probe h (25S) CTCCGCTTATTGATATGC 25S rRNA hybridization Probe 5S GGTCACCCACTACACTACTCGG 5S rRNA hybridization RPL14A +/- 1kb up GGAATTCTCTCCCATATACAAGGAAGG PCR cloning RPL14A RPL14A +/- 1kb dwn CGGATCCCCTACAACCACTGCTGCTGC PCR cloning RPL14A RPL14B+/- 1kb up CGGATCCGGTGGAAGGGAGCTCTTCG PCR verification RPL14B RPL14B +/- 1kb dwn CACATCCCTTCTTTGTTGACG PCR verification RPL14B RPL16A +/- 1kb up GTACCGCCAACCCTTCCAG PCR verification RPL16A RPL16A +/- 1kb dwn CGAACGAACTGTATCGCGG PCR verification RPL16A RPL16B +/- 1kb up AGATAGCGGAGGCAACAAGAG PCR verification RPL16B RPL16B +/- 1kb dwn ACTAGTTCCCTCTTATCTCTC PCR verification RPL16B L14A up DEL GTTTGTCTTAGTGAACAAAGAGAGTCGTGAAAAATAAAAT AAACACGTACGCTGCAGGTCGAC Genomic deletion rpl14A::HIS3MX6 L14A dwn DEL AAATTACATACGAAATATATCAGAATTAAAGATCATGTAC ATATTATCGATGAATTCGAGCTCG Genomic deletion rpl14A::HIS3MX6 L14B up DEL GCATTTAAAGTAACTTCAGGAAATAAACGCGCAAATAAAC CAAAACGTACGCTGCAGGTCGAC Genomic deletion rpl14B::HIS3MX6 L14B dwn DEL TGCTTTCAGTACTTACCTATGGATTTTAGAATTATTCTTC TTTTTATCGATGAATTCGAGCTCG Genomic deletion rpl14A::HIS3MX6 pGAL-L14A up GCGTCGACATGTCCACCGATTCTATTGTC PCR cloning GAL::RPL14A pGAL-L14A dwn GGCGTGTACTTTCACGAC PCR cloning GAL::RPL14A GFP-L14A dwn CGGATCCAGCCTTAGCCAAAGCCTTC PCR cloning RPL14A-yGFP NOC2 up100 CGTGAAGTTAAGGAAGAAAAGGCC Genomic tagging NOC2-TAP-URA3 NOC2 dwn100 GCTTGGAGTGCTCACATGTTAGC Genomic tagging NOG2-TAP-URA3 104 aún no han sido enviadas a degradación. Estas partículas deben acumularse como consecuencia de un punto de control estructural en el estado conformacional erróneo del pre-rRNA 27S como resultado de la ausencia de L14 que impide la asociación de diferentes factores de actuación en trans, especialmente la del factor de exportación Nmd3 [161, 184]. La composición proteica de estas partículas indican que la represión transcripcional de L14 previene el ensamblaje estable de diferentes proteínas ribosómicas, entre ellas se pueden destacar dos grupos de proteínas, primero, aquel formado por las proteínas ribosómicas L17, L39, L26, L37 y L19, todas ellas localizadas alrededor del túnel de salida del polipéptido naciente y predominantemente unidas al dominios I de los rRNAs 25S/5.8S; segundo, el grupo formado por las proteínas ribosómicas L33, L6 y L20, las cuales interaccionan con L14 y se unen al dominio II del rRNA 25S. Interesantemente, L16, que interacciona directamente con L14, L20 y L33 [265], no se ve afectada significativamente por la represión transcripcional de L14. Este resultado sugiere que, a pesar de su íntima interacción, el ensamblaje de L14 y L16 no es interdependiente y que el ensamblaje de L16 es más temprano que el de L14 en las partículas pre-60S. La asociación estable de la mayoría de los factores de actuación en trans perteneciente a la categoría denominada “factores B”, los cuales son importantes para el corte del pre-rRNA 27SB en el sitio C2 se ve afectada negativamente [30, 272]. Este resultado concuerda con el requerimiento de L14 para la maduración del pre-rRNA 27SB. Paradójicamente, el reclutamiento de la mayoría de los factores conocidos como factores A3, los cuales se asocian a las partículas pre-60S de manera interdependiente y están involucrados en el procesamiento del pre-rRNA 27SA3 como también lo está L14 [132], permanecen inalterados o tienen un leve incremento en las partículas pre-60S purificadas tras la represión transcripcional de L14. Este resultado claramente indica que, a diferencia a otras proteínas ribosómicas como L8, cuyo ensamblaje es necesario para la asociación de los factores A3 con las partículas pre-ribosomicas [125, 135], L14 no se requiere para la asociación de estos factores con las partículas pre-60S. De este modo, los defectos en el procesamiento del pre-rRNA 27SA3 observados tras la represión transcripcional de L14 no se deben directamente a la ausencia en la asociación de factores A3 con las partículas pre- 60S. Sin embargo, la represión transcripcional de L14 debe afectar claramente a la función de este conjunto de factores durante la reorganización de la región ITS1/5.8S/ITS2 del prerRNA 27S y el ensamblaje de las proteínas ribosómicas alrededor del túnel de salida que permiten la digestión exonucleolítica 5’-3’ del pre-rRNA 27SA3 al pre-rRNA 27SBS. Nuestros resultados también indican que el defecto en el ensamblaje de las proteínas ribosómicas alrededor del túnel de salida tras la represión transcripcional de L14 no es la consecuencia de la ausencia de factores A3 en las partículas pre-60S, sino el resultado de una formación 105 alterada de sus sitios de unión en el pre-rRNA, defectos que se deben propagar desde la estructura alterada del sitio de unión en el rRNA de L14 tras la represión transcripcional de L14. El procesamiento del pre-rRNA 27SA2 al pre-rRNA 27SA3 no se ve afectado tras la represión transcripcional de L14. De forma consistente, nuestros resultados muestran que L3, la cual es necesaria para este paso [129], está presente en las partículas pre-60S purificadas. Se ha descrito que in vitro el corte endonucleolítico en el sitio A3 por la RNasa MRP solo requiere un sustrato mínimo de RNA de cadena sencilla ([48, 303, 304]). Así la presencia del pre-rRNA 27SA3 en la cepa mutante indica no sólo que el reclutamiento de la RNasa MRP deser efectivo en las condiciones no permisivas sino que este mínimo sustrato requerido deber estar presente en las partículas pre-60S carentes de L14 para asegurar que la RNasa MRP se encuentre activa. De forma general, nuestros resultados concuerdan con resultados previos sobre el ensamblaje de proteínas ribosómicas y la asociación de factores de actuación en trans tras la represión transcripcional de otras proteínas ribosómicas tales como L7, L16, L18, L20, L32 y L33 que se unen al dominio II del rRNA 25S y son vecinas de L14 [125]. La comparación de los resultados de esta publicación con los datos aquí presentados sugiere que el ensamblaje de L14 en partículas tempranas pre-60S es interdependiente con al menos el de L6, L20 y L33. L16 parece que se ensambla más temprano que L14 y mientras su ensamblaje parece ser que se requiere para la incorporación eficiente de L14, lo contrario parece que no es aplicable. La alteración local de la falta de ensamblaje de algunas de estas proteínas en su entorno más cercano en los dominios II y VI (ES39) del rRNA 25S debe afectar negativamente al ensamblaje del segundo conjunto de proteínas (L17, L19, L26 y L39). Es más, estas alteraciones también deben ser las responsables de afectar específicamente a la asociación a partículas pre-60S de un sub-grupo de factores de actuación en trans. Recientemente, se ha resuelto la estructura de partículas pre-60S nucleoplásmicas tardías en levadura a alta resolución por cryo-EM [147, 153, 247] pero desafortunadamente ninguna estructura está disponible para partículas pre-60S tempranas hasta el momento. De este modo, carecemos de recursos moleculares para explicar la lógica para estos efectos específicos en la asociación de estos distintos factores. Finalmente, el hecho de que la represión transcripcional ciertas proteínas ribosómicas tales como L14, L6, L20 y L33 cause entre otras cosas la perdida de estas proteínas de manera conjunta de partículas pre-60S explicando de una manera lógica y sencilla por qué los fenotipos de procesamiento del pre-rRNA detectados tras la represión transcripcional de todas estas proteínas son prácticamente idénticas. En esta Tesis, también hemos estudiado el efecto de la truncación de la extensión C- terminal de la proteína ribosómica L16 sobre la viabilidad celular, el procesamiento de los 106 pre-rRNAs y la maduración de la subunidad 60S. De las diferentes truncaciones estudiadas, sólo aquella que implica los últimos 18 aminoácidos de L16B (L16B-N180) fue capaz de permitir el crecimiento, como única fuente de L16 in vivo. Nuestros resultados también muestran que, independientemente de su impacto en el crecimiento celular, todas las variantes truncadas de L16B se expresan y son capaces de ensamblarse en partículas pre- 60S. Este resultado es compatible con resultados obtenidos para un estudio similar de la extensión C-terminal de la proteína ribosómica L13a de mamíferos, que indican que la extensión C-terminal de L13a no está activamente involucrada en la unión al rRNA o en el ensamblaje a los ribosomas [190]. No obstante, las partículas pre-60S que contienen truncaciones de L16B no viables son claramente defectuosas, ya que en su mayoría fueron retenidas en el nucléolo. Por el contrario, no hemos observado defectos en el procesamiento del pre-rRNA para el mutante viable L16B-N180. Sin embargo, si encontramos defectos en el procesamiento del pre-rRNA para los mutantes truncados no viables. Los fenotipos observados fueron menos pronunciados que los observados tras la represión transcripcional de L16. Por tanto el ensamblaje del dominio globular de L16 debe ser suficiente para promover algún tipo de plegamiento del pre-rRNA 27SA que resulta en una mayor estabilidad de las partículas tempranas pre-60S. Estos resultados coinciden con resultados publicados de manera independiente por el del Prof. Milikereit y colaboradores donde se evalua el impacto de dos truncaciones de la extensión C-terminal de L16, una truncación idéntica a la L16B-N147 y otra similar a L16B-N168 [192]. No se conoce porque la eliminación de los últimos 18 aminoácidos de L16B prácticamente no presenta ningún efecto sobre la producción de subunidades 60S mientras que truncaciones más grandes de la proteína daña fuertemente la biogénesis. Nosotros especulamos que, mientras que en el mutante rpl16-N180 no afecta a la interacción entre L14-L16, por otro lado en los mutantes rpl16-N168 y rpl16-N147 no permite una eficiente interacción de ambas proteínas ribosómicas, provocando como consecuencia mal plegamiento del rRNA durante el ensamblaje de las subunidades 60S. Por otro lado, en esta Tesis, hemos estudiado la relación funcional existente entre la extensión C-terminal especifica de eucariotas de L14 y L16 [10, 11]. Para ello, hemos coexpresado la proteína L14A-N123 y la proteína L16B-N180 como fuentes de L14 y L16, respectivamente, en células de levadura. El alelo rpl14A-N123 que carece de los últimos 16 aminoácidos de L14A confiere un defecto leve en la biogénesis de 60S que afecta sutilmente al crecimiento a 30 ºC cuando es expresado como única fuente de L14 desde un plásmido centromérico [176]. 107 Nuestros resultados indican que, aunque esta combinación no produce ningún defecto sinérgico en condiciones normales de crecimiento (medio YPD, 30 ºC), no permite el crecimiento en medio de cultivo que contiene una concentración sub-letal de paromomicina. Este resultado sugiere que la interacción completa de las extensiones C-terminales de L14 y L16 no es estrictamente necesaria para el ensamblaje de subunidades 60S pero es al menos necesaria para algunos aspectos funcionales de las subunidades 60S. En conclusión, en esta Tesis se demuestra que tanto L16 y L14 juegan un papel esencial en las etapas tempranas del ensamblaje de las subunidades 60S, más específicamente, durante el procesamiento del pre-rRNA 27SA3 y la formación de los precursores del pre-rRNA 27SB. El modo en que L16 lleva a cabo este papel aún no está claro. El laboratorio del Prof. Kressler ha demostrado que una variante truncada de la proteína ribosómica L14A, la cual carece de gran parte de su α-hélice C-terminal, confiere defectos en crecimiento y muestra una interacción sintético letal con distintos alelos de MAK5, que codifican para una helicasa de RNA involucrada en la biogénesis de la subunidad 60S, EBP2 y NOP16 [176, 305]. De este modo se ha sugerido que Mak5 podría requerirse para el correcto ensamblaje de L14 y sus vecinos en partículas tempranas pre- 60S [176]. De igual modo, en Escherichia coli, se ha demostrado que la helicasa de RNA SrmB es requerida para el correcto ensamblaje de la proteína ribosómica L13, la cual es la ortóloga bacteriana de la proteína ribosómica L16 de S. cerevisiae, y posteriormente ensamblando otras proteínas ribosómicas [187, 306]. La realización de más experimentos deberían permitirnos esclarecer si, de forma similar a L14, el ensamblaje de L16 también es facilitado por la actividad de Mak5 y/o proteínas con las que interacciona genéticamente y cómo, si es el caso, contribuye la extensión c-terminal especifica de eucariotas a este ensamblaje. A pesar del hecho de que L14 y L16 interaccionan íntimamente por sus extensiones C-terminales, el ensamblaje de L16 parece ser que precede al de L14 y no es estrictamente dependiente de estas extensiones. 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