The Beak of Eukaryotic Ribosomes: Life, Work and Miracles

Citation: Martín-Villanueva, S.; Galmozzi, C.V.; Ruger-Herreros, C.; Kressler, D.; de la Cruz, J. The Beak of Eukaryotic Ribosomes: Life, Work and Miracles. Biomolecules 2024,14, 882. https://doi.org/10.3390/ biom14070882 Academic Editor: Leonard B. Maggi Jr. Received: 19 June 2024 Revised: 19 July 2024 Accepted: 21 July 2024 Published: 22 July 2024 Copyright: © 2024 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/). biomolecules Review The Beak of Eukaryotic Ribosomes: Life, Work and Miracles Sara Martín-Villanueva 1,2,† , Carla V. Galmozzi 1,2,† , Carmen Ruger-Herreros 1,2 , Dieter Kressler 3 and Jesús de la Cruz 1,2,* 1Instituto de Biomedicina de Sevilla, Hospital Universitario Virgen del Rocío/CSIC/Universidad de Sevilla, E-41013 Seville, Spain; [email protected] (S.M.-V.); [email protected] (C.V.G.); [email protected] (C.R.-H.) 2Departamento de Genética, Facultad de Biología, Universidad de Sevilla, E-41012 Seville, Spain 3Department of Biology, University of Fribourg, CH-1700 Fribourg, Switzerland; [email protected] *Correspondence: [email protected]; Tel.: +34-955-923-126 †These authors contributed equally to this work. Abstract: Ribosomes are not totally globular machines. Instead, they comprise prominent structural protrusions and a myriad of tentacle-like projections, which are frequently made up of ribosomal RNA expansion segments and N- or C-terminal extensions of ribosomal proteins. This is more evident in higher eukaryotic ribosomes. One of the most characteristic protrusions, present in small ribosomal subunits in all three domains of life, is the so-called beak, which is relevant for the function and regulation of the ribosome’s activities. During evolution, the beak has transitioned from an all ribosomal RNA structure (helix h33 in 16S rRNA) in bacteria, to an arrangement formed by three ribosomal proteins, eS10, eS12 and eS31, and a smaller h33 ribosomal RNA in eukaryotes. In this review, we describe the different structural and functional properties of the eukaryotic beak. We discuss the state-of-the-art concerning its composition and functional significance, including other processes apparently not related to translation, and the dynamics of its assembly in yeast and human cells. Moreover, we outline the current view about the relevance of the beak’s components in human diseases, especially in ribosomopathies and cancer. Keywords: ribosomal protuberances; eS12; eS31; Ubi3; eS10; translation accuracy; ribosome biogenesis 1. Introduction: Protuberances of the Ribosomal Subunits Ribosomes are intricate molecular machinery found in the cytoplasm of all organisms. They play crucial roles during the translation of cellular mRNAs by efficiently and accurately decoding their genetic information. In addition, ribosomes can be found inside two types of organelles, chloroplasts and mitochondria [1–4]. All ribosomes are universally composed of two subunits, the small and large ribosomal subunits (r-subunits), which comprise ribosomal RNAs (rRNAs) and ribosomal proteins (rproteins). The large r-subunit is about twice the size of the small r-subunit [ 2 , 5 ]. In a model eukaryote, such as the yeast Saccharomyces cerevisiae, the small r-subunit or 40S is composed of a single 18S rRNA and 33 different r-proteins; in turn, the large r-subunit or 60S contains three rRNAs (5S, 5.8S and 25S rRNAs) and 46 (47 in humans) r-proteins [6,7]. The general shape of the ribosomes has been known since the early 1970s; however, structures at high resolution have only been obtained since the beginning of the twenty-first century, following significant advances in structural technologies, such as X-ray crystallography and cryo-electron microscopy (cryo-EM) (e.g., [ 7 – 10 ]). In agreement with the functional conservation of ribosomes in all organisms [ 2 , 5 , 11 , 12 ], ribosomes display considerable structural similarity in prokaryotes, organelles and eukaryotes, although organellar ribosomes have substantially diverged from bacterial ones, with which they share a common ancestor (e.g., [ 3 , 13 ]). Moreover, structural studies have clearly shown that eukaryotic ribosomes Biomolecules 2024,14, 882. https://doi.org/10.3390/biom14070882 https://www.mdpi.com/journal/biomolecules Biomolecules 2024,14, 882 2 of 27 (25–30 nm in diameter) are larger and more complex than their prokaryotic (20 nm in diameter) and organellar counterparts [ 2 , 3 , 5 – 7 ]. When observed at low resolution, ribosomes appear flattened and spherical in shape; however, this observation is far from being correct. In fact, both r-subunits are extremely irregular complexes containing different domains and exhibiting different discernible protuberances. The small r-subunit is formed by four different structural domains, the head, the platform, the body and the beak; the beak domain is the main protrusion found in small r-subunits (Figure 1A). The large r-subunit, which is a more compact unit, displays three characteristic protuberances, the L1- and the P-stalk and the central protuberance (Figure 1B). Importantly, the structure of several r-subunit protuberances and protrusions has only been determined at high resolution in a few organisms, a fact that is most likely due to the high mobility of these regions, which is clearly connected to their functions (e.g., [ 6 ]). Thus, the movement of the beak as part of the head of 40S r-subunits is important to allow the loading of mRNA on the ribosome and the interaction with translation factors, as we will discuss in detail later on. In turn, the primary role of the P-stalk is to provide a flexible hook outside of the core of the ribosome to recruit and then stimulate the activity of different translational GTPases during various stages of translation (e.g., [ 14 – 16 ]). The P-stalk also enables the activation of the Gcn2 kinase and mediates the interaction with distinct ribosome-inactivating proteins (e.g., [ 17 – 20 ]). The L1-stalk acts as a flexible protuberance at the antipodes of the P-stalk, facilitating the binding, movement and release of the deacylated tRNAs [21,22]. Biomolecules 2024, 14, 882 2 of 28 eukaryotic ribosomes (25–30 nm in diameter) are larger and more complex than their prokaryotic (20 nm in diameter) and organellar counterparts [2,3,5–7]. When observed at low resolution, ribosomes appear flattened and spherical in shape; however, this observation is far from being correct. In fact, both r-subunits are extremely irregular complexes containing different domains and exhibiting different discernible protuberances. The small rsubunit is formed by four different structural domains, the head, the platform, the body and the beak; the beak domain is the main protrusion found in small r-subunits (Figure 1A). The large r-subunit, which is a more compact unit, displays three characteristic protuberances, the L1- and the P-stalk and the central protuberance (Figure 1B). Importantly, the structure of several r-subunit protuberances and protrusions has only been determined at high resolution in a few organisms, a fact that is most likely due to the high mobility of these regions, which is clearly connected to their functions (e.g., [6]). Thus, the movement of the beak as part of the head of 40S r-subunits is important to allow the loading of mRNA on the ribosome and the interaction with translation factors, as we will discuss in detail later on. In turn, the primary role of the P-stalk is to provide a flexible hook outside of the core of the ribosome to recruit and then stimulate the activity of different translational GTPases during various stages of translation (e.g., [14–16]). The P-stalk also enables the activation of the Gcn2 kinase and mediates the interaction with distinct ribosome-inactivating proteins (e.g., [17–20]). The L1-stalk acts as a flexible protuberance at the antipodes of the P-stalk, facilitating the binding, movement and release of the deacylated tRNAs [21,22]. Figure 1. Protuberances of the r-subunits. (A) Structure of the mature 40S r-subunit of Saccharomyces cerevisiae showing its different structural domains (head, body, platform and beak) and highlighting the beak elements that include helix h33 of the 18S rRNA and the r-proteins eS10, eS12 and eS31. The 40S r-subunit is shown from the interface view; 18S rRNA is colored in gray (except h33 in yellow) and r-proteins not belonging to the beak structure are colored in green. Protein Data Bank (PDB) code: 4V88. (B) Structure of the mature 60S r-subunit of S. cerevisiae showing its different substructures (L1 and P0 stalks, and central protuberance) and highlighting the different protuberances: L1-stalk formed by helices H76-78 of 25S rRNA and the r-protein uL1; central protuberance formed by 5S rRNA and r-proteins uL5 and uL18; P0-stalk formed by helices H43-44 of 25S rRNA and the r-proteins uL11, uL10 (P0), P1 and P2 (two copies each). As other examples of protrusions found in the 60S r-subunits, we highlight helices H38 and H69 (yellow) and the long extensions of the r-proteins eL19 and eL24 (purple). The 60S r-subunit is shown from the interface view; 25S and Figure 1. Protuberances of the r-subunits. (A) Structure of the mature 40S r-subunit of Saccharomyces cerevisiae showing its different structural domains (head, body, platform and beak) and highlighting the beak elements that include helix h33 of the 18S rRNA and the r-proteins eS10, eS12 and eS31. The 40S r-subunit is shown from the interface view; 18S rRNA is colored in gray (except h33 in yellow) and r-proteins not belonging to the beak structure are colored in green. Protein Data Bank (PDB) code: 4V88. (B) Structure of the mature 60S r-subunit of S. cerevisiae showing its different substructures (L1 and P0 stalks, and central protuberance) and highlighting the different protuberances: L1-stalk formed by helices H76-78 of 25S rRNA and the r-protein uL1; central protuberance formed by 5S rRNA and r-proteins uL5 and uL18; P0-stalk formed by helices H43-44 of 25S rRNA and the r-proteins uL11, uL10 (P0), P1 and P2 (two copies each). As other examples of protrusions found in the 60S r-subunits, we highlight helices H38 and H69 (yellow) and the long extensions of the r-proteins eL19 and eL24 (purple). The 60S r-subunit is shown from the interface view; 25S and 5.8S rRNAs are colored in gray and the rest of the r-proteins are colored in light blue. PDB code: 6OIG. Cartoons were generated using UCSF Chimera (https://www.rbvi.ucsf.edu/chimera; accessed on 1 June 2024). Biomolecules 2024,14, 882 3 of 27 In addition to being part of the prominent, above-described protuberances, many r-proteins have long non-globular extensions, typically serpentine ones that become deeply embedded inside the rRNA core of the r-subunits and stabilize their overall structure. There are also r-protein extensions (see Figure 1B for examples) that project out of the ribosome and may serve as additional regions for interactions of the ribosome with factors or cellular structures [ 23 – 25 ]. Frequently, these extensions, which are located at the N- and/or C-terminal ends of r-proteins, are not well ordered and are highly mobile due to the lack of stable interactions with the core of the ribosome; consequently, it has not been possible to model all of them from the current cryo-EM density maps. Protuberances are not only flexible components of ribosomes, but also dynamic structures. The eukaryotic P-stalk is a pentameric complex, composed of the essential r-protein uL10 (previously named P0) and two non-essential heterodimers made up of another two r-proteins (named P1 and P2), that is connected to the body of the 60S r-subunit by the uL11 r-protein [ 15 , 26 ]. P1 and P2, when phosphorylated, can exchange during translation with their equivalent cytoplasmic pool of non-phosphorylated P1 and P2 variants [ 27 – 29 ]. Interestingly, in yeast, P1 and P2 r-proteins are not essential for cell viability, but their absence affects differently the translation of specific mRNAs [ 15 , 30 ]. Furthermore, it has been described that the amount of P1/P2 r-proteins bound to the ribosome changes under different physiological conditions. For instance, when yeast cells enter the stationary phase, the P1/P2 r-proteins are practically absent from ribosomes [ 31 ]. This and other observations made in different eukaryotes (e.g., [ 32 , 33 ]) have allowed different authors to propose that the eukaryotic P-stalk acts as a regulator of translation and that changes in its composition could selectively control the translation of specific groups of mRNAs [ 34 ]. In agreement with a regulatory role of the P-stalk during translation, mutation of the phosphorylation sites of the yeast acidic P1/P2 r-proteins does not alter their interaction with the ribosome but influences the translation of specific mRNAs, among them, those related to osmotic stress [ 35 ]. Further experiments, using high-resolution techniques (e.g., ribosome profiling), are required to provide details on such a regulatory process and on the specific proteome translated upon changes in P-stalk abundance and phosphorylation status. To the best of our knowledge, aside from the acidic P1/P2 r-proteins, there is no evidence of exchangeability of r-proteins from the other ribosomal protuberances, including the beak; however, there are interesting reports about several other r-proteins whose nascent forms can replace an older copy of themselves on a mature ribosome. (i) Thus, uL16 has been described as an r-protein potentially able to cycle on and off large r-subunits [36–38] . uL16 is strategically positioned on the surface of the evolutionarily conserved core of the 60S r-subunit, near the corridor through which aminoacyl-tRNAs move during accommodation and also near other functional centers, such as the GTPase-associated center (GAC) and the peptidyl transferase center (PTC). uL16 is required for the joining of r-subunits during translation initiation and the rotation status of the ribosome [ 39 – 41 ]; importantly, the availability to assemble uL16 onto large r-subunits could also be used as a translational regulatory mechanism to limit global translation under unfavorable circumstances [ 37 , 41 ]. (ii) Another interesting r-protein is RACK1 (Asc1 in yeast). RACK1 is a WD40-domain protein located at the head region of the 40S r-subunit near the mRNA exit site, where it interacts with other r-proteins, among them uS3, several kinases, and translation initiation factors [ 42 , 43 ]. RACK1 has an important role in different aspects of the translation process: it is required for efficient translation of mRNAs with short open reading frames (e.g., those of r-protein genes), it is critical for translation during heat stress, and it facilitates ribosome-associated quality control (RQC) mechanisms such as those that are involved in the rescue of stalled collided ribosomes at consecutive rare codons, such as CGA (Arg) in yeast [ 44 – 49 ]. Although there is no experimental demonstration for RACK1 to cycle on and off ribosomes [ 50 ], it is clear that RACK1 is a non-essential r-protein whose loss does not disrupt ribosome integrity and translation [ 46 ]. Moreover, as RACK1 protein levels can be modulated by a variety of environmental insults, such as hypoxic stress, glucose deprivation or amino acid starvation or by the physiological cellular status (exponential versus Biomolecules 2024,14, 882 4 of 27 stationary growth phase) (e.g., [ 51 ]), it is possible that the RACK1’s ribosome association could be regulated, thereby, promoting differential translation. (iii) Another important inductor of exchangeability is chemical damage. Originally reported in prokaryotes [ 52 ], yeast ribosomes containing chemically damaged r-proteins can also be repaired by exchanging these with undamaged r-proteins, as convincingly demonstrated by the Karbstein laboratory [ 38 , 53 , 54 ]. The repair of damaged ribosomes might represent an important mechanism for maintaining the translational activity of cells following different insults as, for example, those produced by oxidative stress [ 53 , 55 ]. An analogous repair process via protein replacement has also been suggested to occur in neurons; thus, implying that this mechanism has been evolutionarily conserved (e.g., [ 56 ]). In yeast, the molecular details for a ribosome repair mechanism have been provided upon oxidation of eS26 and uL16, which are released from damaged ribosomes by their respective dedicated chaperones, Tsr2 and Sqt1, generating transiently eS26- and uL16-deficient ribosomes that are subsequently repaired with newly made r-proteins [ 38 ]. Ribosomes lacking eS26 can also be generated in a Tsr2-dependent manner upon the exposure of yeast cells to high salt or high pH conditions. Ribosome repair is extremely relevant from the physiological point of view as eS26-lacking ribosomes preferentially translate specific transcripts bearing Kozak sequence variations, including mRNAs enabling the biological response to high salt and high pH insults [ 55 ]. The recovery from stress is concomitant to the reincorporation of eS26 into ribosomes, again in a Tsr2-dependent manner [ 38 , 53 ]. This sophisticated system of autoregulation resembles that previously reported for the translation circuit of leaderless mRNAs (lmRNAs) in bacteria. These lmRNAs can be generated in response to adverse environmental conditions, some of them (e.g., the presence of antibiotics such as kasugamycin in the culture media) also being able to reprogram ribosomes to translate preferentially lmRNAs. Interestingly, this reprogramming involves the formation of stable r-particles (referred to as 61S particles) deficient in almost a dozen r-proteins from the small r-subunit, among them bS1 and other r-proteins associated along the path of the mRNA through this r-subunit [57,58]. Of special attention is the central protuberance (CP), where the 5S ribonucleoprotein particle (RNP), which is composed of 5S rRNA and r-proteins uL5 and uL18, plays an important regulatory role (Figure 1B). The whole 5S RNP, rather than its individual components, is incorporated as a prefabricated complex into early pre-60S r-particles during the nucleolar ribosome biogenesis phase, and it temporally adopts a conformation that is different from the one in the mature 60S r-subunit (e.g., [ 59 – 61 ]). From yeast to humans, ribosome biogenesis is tightly coupled to cell growth and proliferation, with the assembly of the 5S RNP playing a central regulatory role. Thus, in yeast, an imbalanced production of rRNAs and r-proteins generates defects in ribosome biogenesis leading to the accumulation of ribosome-unbound uL18, likely as part of the 5S RNP, which induces a delay in the G1/S phase of the cell cycle [ 62 ]. This behavior has been interpreted as part of a protective mechanism that prevents cell cycle progression when ribosome biogenesis is impaired, i.e., when not all necessary components are sufficiently available to ensure a complete and satisfactory assembly of ribosomes. In metazoans, the 5S RNP clearly accumulates when ribosome biogenesis is impaired [ 63 – 66 ]. The free 5S RNP binds to MDM2 (HDM2 in humans), which is an E3 ubiquitin ligase that ubiquitinates p53 and thereby channels it to degradation via the proteasome. Upon binding of the 5S RNP to MDM2, which occurs mutually exclusively to its binding to pre-60S r-particles, p53 escapes from MDM2-mediated degradation and accumulates [ 67 , 68 ]. Concomitantly, p53 is activated and, thus, exerts its different anti-proliferative functions, ranging from temporary cell cycle arrest to apoptosis [ 69 ]. The implications of the involvement of ribosome biogenesis in the regulation of p53 in human health and disease have been extensively discussed in other reviews (e.g., [68,70–72]) and will also be examined later in this work. This review is aimed at giving insights into the composition, structure, role, biogenesis and dysfunction of the components of the beak, which is the most prominent protuberance found in all small r-subunits and so-called because of its resemblance to a bird’s beak. We discuss all these features of the eukaryotic beak by specially focusing on the current Biomolecules 2024,14, 882 5 of 27 knowledge about the beak of 40S r-subunits from the yeast S. cerevisiae and by highlighting similarities and differences compared to the beak of human ribosomes. 2. Composition of the Beak of the Eukaryotic Ribosome Despite the fact that the beak is overall an easily recognizable structure in the small r-subunits of all ribosomes, the composition of the beak is quite different within the three domains of life. In bacteria, the beak is composed exclusively of rRNA, specifically, the helix h33 of 16S rRNA [ 9 , 73 , 74 ]. In contrast, the beak of eukaryotic ribosomes has been transformed into a mixture of rRNA and specific r-proteins not found in bacteria, with the biological reasons for this transformation remaining unsolved. The r-proteins bound to helix h33 of eukaryotic 18S rRNA, which itself is shorter than the bacterial h33, are eS10, eS12 and eS31 [ 1 , 2 , 6 ]. It is accepted that the structural core components of the archaeal ribosomes are of prokaryotic origin, to which specific elements, some shared with eukaryotes, have been added; therefore, archaeal ribosomes represent intermediate steps towards the evolution of eukaryotic ribosomes [ 75 ]. In this sense, it is interesting to mention that the beak of archaeal small r-subunits has a transitional complexity from an allrRNA to an rRNA/r-protein protrusion; accordingly, many archaeal genomes encode clear homologs of the eukaryotic eS31 r-protein, which in all cases, however, lack the eukaryotespecific N-terminal extension [ 76 , 77 ]. In addition, cryo-EM has shown that ribosomes of distinct archaea contain at least two copies of eL8, one at the canonical location on the large r-subunit and another one bound at a position on h33 that is equivalent to the one occupied by eS12 on the eukaryotic beak [ 78 ]; further predictions suggest that this feature, i.e., the presence of eL8 in the beak, occurs in all archaeal ribosomes [ 78 ]. Moreover, this observation suggests that eS12 evolved from eL8, as both proteins share conserved regions and belong to the same family (InterPro entry IPR004038). Finally, it is clear that eukaryotic eS10 has no counterpart in archaeal ribosomes, as evidenced by different database searches, including BLAST [ 77 , 79 ]. A comparison of the beak composition and structure of ribosomes from prototypical bacteria, archaea and eukaryotes is shown in Figure 2. Ribosomes present in organelles also contain all the structural landmarks that are characteristic of cytoplasmic ribosomes of prokaryotes and eukaryotes, including the beak. However, mito- and chlororibosomes have been found to be extremely diverse in terms of their composition, including the acquisition of organelle-specific r-proteins that has an impact on their overall structures. In general, chlororibosomes resemble bacterial ribosomes [ 80 – 82 ]; hence, the beak of these ribosomes is formed by the h33 rRNA protrusion and is devoid of r-proteins (Figure 3). In marked contrast to chlororibosomes, the characterization of mitoribosomes from diverse species representing the different major groups of eukaryotes has revealed that these have diverged considerably from each other and from prokaryotic and eukaryotic ribosomes [ 3 , 83 , 84 ]. In mitoribosomes, the beak can vary from the all-RNA prototype found in yeast to the massive protein-based beak found in the kinetoplastid Trypanosoma brucei (Figure 3). Cytoplasmic ribosomes of the yeast S. cerevisiae contain a standard beak, composed of a helix h33 of 52 nucleotides and a single copy of three r-proteins, eS10, eS12 and eS31 (Figure 4) [ 6 ]. Yeast eS10 is encoded by two paralogous genes, RPS10A (YOR293W) and RPS10B (YMR230W). The two genes code for the virtually identical eS10A and eS10B r-proteins of 105 amino acids and ca. 12.7 kDa that only differ in three solvent-exposed amino acids (E6, D7 and T98 in eS10A versus Q6, E7 and S98 in eS10B). Mutants harboring individual deletions of the RPS10A and RPS10B genes are viable in different yeast backgrounds; moreover, while the rps10B ∆ null mutant grows practically identical to the wild-type strain, the rps10A ∆ null mutant exhibits only a mild increase in the doubling time [ 85 ]. In all genetic backgrounds, eS10 is an essential protein as the rps10A ∆ rps10B ∆ double mutant is inviable [85,86]. Yeast eS10 is a mostly globular protein; however, it contains an unstructured C-terminal extension of about 20 amino acids, which interacts with uS3 at the base of the beak in the mRNA entry channel [ 87 ]. In contrast to eS10 and most yeast r-proteins, eS12 and eS31 are non-essential r-proteins that are encoded by single-copy Biomolecules 2024,14, 882 6 of 27 genes (RPS12 or YOR369C, and RPS31 or UBI3 or YLR167W, respectively). However, in most genetic backgrounds, both the rps12 ∆ and the ubi3 ∆ mutant display a severe growth impairment [ 85 , 88 , 89 ]. Yeast eS12 is a small globular protein of 143 amino acids and ca. 15.5 kDa, containing an unstructured N-terminal extension of around 25 amino acids whose deletion causes a slow growth phenotype of still uncertain significance (our unpublished results). On the other hand, yeast eS31 is a small r-protein of 76 amino acids consisting of a globular domain, which is well conserved from archaea to eukaryotes, and a eukaryotespecific N-terminal extension of about 25 amino acids, which extends toward the ribosomal A-site and has relevant functions in translation and small r-subunit assembly ([ 1 , 76 , 90 ]; see later). More interestingly, it has been well reported that in most eukaryotes eS31 as well as eL40 are produced as C-terminal parts of ubiquitin-fused precursor proteins, which are rapidly processed to individual ubiquitin and r-protein moieties before assembly of the corresponding r-protein into the small and large r-subunit, respectively [ 91 ]. The biological relevance of maintaining these fusions during evolution for the correct production and assembly of these r-proteins and for the possible co-regulation of two related cellular functions, protein synthesis and protein degradation, has been previously covered and will not be further discussed in this review [91,92]. Biomolecules 2024, 14, 882 6 of 28 Figure 2. Comparison of small r-subunits of bacteria, archaea and eukaryotes. (A) Small r-subunit of Escherichia coli; PDB code: 7OE1; (B) small r-subunit of Thermus thermophilus; PDB code: 1J5E; (C) small r-subunit of Pyrococcus abyssi; PDB code: 7ZHG; (D) small r-subunit of S. cerevisiae; PDB code: 4V88; (E) small r-subunit of Homo sapiens; PDB code: 7R4X. In all cases, the interface view of the individual r-subunits is shown. The rRNA is colored in gray and the r-proteins in green. The beak (b) of all r-subunits is highlighted; helix h33 of rRNA is colored in yellow, and the r-proteins eS31, eL8, eS10 and eS12 in the indicated colors. The r-protein uS3, which is located at the base of the beak, is colored in pink. Cartoons were generated using UCSF Chimera (https://www.rbvi.ucsf.edu/chimera; accessed on 1 June 2024). Ribosomes present in organelles also contain all the structural landmarks that are characteristic of cytoplasmic ribosomes of prokaryotes and eukaryotes, including the beak. However, mito- and chlororibosomes have been found to be extremely diverse in terms of their composition, including the acquisition of organelle-specific r-proteins that has an impact on their overall structures. In general, chlororibosomes resemble bacterial ribosomes [80–82]; hence, the beak of these ribosomes is formed by the h33 rRNA protrusion and is devoid of r-proteins (Figure 3). In marked contrast to chlororibosomes, the characterization of mitoribosomes from diverse species representing the different major groups of eukaryotes has revealed that these have diverged considerably from each other and from prokaryotic and eukaryotic ribosomes [3,83,84]. In mitoribosomes, the beak can vary from the all-RNA prototype found in yeast to the massive protein-based beak found in the kinetoplastid Trypanosoma brucei (Figure 3). Figure 2. Comparison of small r-subunits of bacteria, archaea and eukaryotes. (A) Small r-subunit of Escherichia coli; PDB code: 7OE1; (B) small r-subunit of Thermus thermophilus; PDB code: 1J5E; (C) small r-subunit of Pyrococcus abyssi; PDB code: 7ZHG; (D) small r-subunit of S. cerevisiae; PDB code: 4V88; (E) small r-subunit of Homo sapiens; PDB code: 7R4X. In all cases, the interface view of the individual r-subunits is shown. The rRNA is colored in gray and the r-proteins in green. The beak (b) of all r-subunits is highlighted; helix h33 of rRNA is colored in yellow, and the r-proteins eS31, eL8, eS10 and eS12 in the indicated colors. The r-protein uS3, which is located at the base of the beak, is colored in pink. Cartoons were generated using UCSF Chimera (https://www.rbvi.ucsf.edu/chimera; accessed on 1 June 2024). Biomolecules 2024,14, 882 7 of 27 Biomolecules 2024, 14, 882 7 of 28 Figure 3. Comparison of the structural features of the small subunit of the mitochondrial ribosome from S. cerevisiae (A) and Trypanosoma brucei (B), and of the chloroplast ribosome from Spinacia oleracea (C). The PDB codes are 5MRC, 6HIW, and 5MMJ, respectively. In all cases, the interface view of the individual r-subunits is shown. The rRNA is colored in gray and the r-proteins in green. The beak (b) of all r-subunits is highlighted; helix h33 of rRNA is colored in yellow, and the set of specific r-proteins present in the T. brucei small r-subunit are shown in different colors. Cartoons were generated using UCSF Chimera (https://www.rbvi.ucsf.edu/chimera; accessed on 1 June 2024). Cytoplasmic ribosomes of the yeast S. cerevisiae contain a standard beak, composed of a helix h33 of 52 nucleotides and a single copy of three r-proteins, eS10, eS12 and eS31 (Figure 4) [6]. Yeast eS10 is encoded by two paralogous genes, RPS10A (YOR293W) and RPS10B (YMR230W). The two genes code for the virtually identical eS10A and eS10B rproteins of 105 amino acids and ca. 12.7 kDa that only differ in three solvent-exposed amino acids (E6, D7 and T98 in eS10A versus Q6, E7 and S98 in eS10B). Mutants harboring individual deletions of the RPS10A and RPS10B genes are viable in different yeast backgrounds; moreover, while the rps10B∆ null mutant grows practically identical to the wildtype strain, the rps10A∆ null mutant exhibits only a mild increase in the doubling time [85]. In all genetic backgrounds, eS10 is an essential protein as the rps10A∆ rps10B∆ double mutant is inviable [85,86]. Yeast eS10 is a mostly globular protein; however, it contains an unstructured C-terminal extension of about 20 amino acids, which interacts with uS3 at the base of the beak in the mRNA entry channel [87]. In contrast to eS10 and most yeast rproteins, eS12 and eS31 are non-essential r-proteins that are encoded by single-copy genes (RPS12 or YOR369C, and RPS31 or UBI3 or YLR167W, respectively). However, in most genetic backgrounds, both the rps12∆ and the ubi3∆ mutant display a severe growth impairment [85,88,89]. Yeast eS12 is a small globular protein of 143 amino acids and ca. 15.5 kDa, containing an unstructured N-terminal extension of around 25 amino acids whose deletion causes a slow growth phenotype of still uncertain significance (our unpublished results). On the other hand, yeast eS31 is a small r-protein of 76 amino acids consisting of a globular domain, which is well conserved from archaea to eukaryotes, and a eukaryotespecific N-terminal extension of about 25 amino acids, which extends toward the ribosomal A-site and has relevant functions in translation and small r-subunit assembly ([1,76,90]; see later). More interestingly, it has been well reported that in most eukaryotes eS31 as well as eL40 are produced as C-terminal parts of ubiquitin-fused precursor proteins, which are rapidly processed to individual ubiquitin and r-protein moieties before assembly of the corresponding r-protein into the small and large r-subunit, respectively [91]. The biological relevance of maintaining these fusions during evolution for the correct Figure 3. Comparison of the structural features of the small subunit of the mitochondrial ribosome from S. cerevisiae (A) and Trypanosoma brucei (B), and of the chloroplast ribosome from Spinacia oleracea (C). The PDB codes are 5MRC, 6HIW, and 5MMJ, respectively. In all cases, the interface view of the individual r-subunits is shown. The rRNA is colored in gray and the r-proteins in green. The beak (b) of all r-subunits is highlighted; helix h33 of rRNA is colored in yellow, and the set of specific r-proteins present in the T. brucei small r-subunit are shown in different colors. Cartoons were generated using UCSF Chimera (https://www.rbvi.ucsf.edu/chimera; accessed on 1 June 2024). Biomolecules 2024, 14, 882 8 of 28 production and assembly of these r-proteins and for the possible co-regulation of two related cellular functions, protein synthesis and protein degradation, has been previously covered and will not be further discussed in this review [91,92]. Figure 4. The r-proteins from the beak (b) of the yeast 40S r-subunit and the equivalent ones from the beak of the Pyrococcus abyssi 30S r-subunit. (A) Structure of the yeast 40S r-subunit (PDB code 4V88). (B) Structure of yeast eS12, eS31 and eS10 r-proteins are shown as found in the 40S r-subunit before or after 90° rotation on the Y-axis. (C) Structure of the equivalent r-proteins eL8 and eS31 from P. abyssi are also shown as found in the 30S r-subunit (PDB code 7ZHG). Note that the first 19 N-terminal residues of yeast eS12, the six first residues of yeast eS31, the last nine C-terminal residues of yeast eS10, the first residue of P. abyssi eL8 and the first and the last residue of P. abyssi eS31 are not present in the structures. Cartoons were generated using UCSF Chimera (https://www.rbvi.ucsf.edu/chimera; accessed on 1 June 2024). In consonance with the critical importance of yeast eS10, eS12 and eS31 r-proteins for cell growth, it has also been reported that loss-of-function mutations in the genes coding for these proteins in other model eukaryotes (Caenorhabditis elegans, Drosophila melanogaster, Danio rerio, Mus musculus, Homo sapiens) lead to a myriad of adverse phenotypes, including lethality, increased cell death, cell cycle arrest, reduced fertility, organ development defects and tumorigenesis [93]. Figure 4. The r-proteins from the beak (b) of the yeast 40S r-subunit and the equivalent ones from the beak of the Pyrococcus abyssi 30S r-subunit. (A) Structure of the yeast 40S r-subunit (PDB code 4V88). (B) Structure of yeast eS12, eS31 and eS10 r-proteins are shown as found in the 40S r-subunit before or after 90 ◦ rotation on the Y-axis. (C) Structure of the equivalent r-proteins eL8 and eS31 from P. abyssi are also shown as found in the 30S r-subunit (PDB code 7ZHG). Note that the first 19 N-terminal residues of yeast eS12, the six first residues of yeast eS31, the last nine C-terminal residues of yeast eS10, the first residue of P. abyssi eL8 and the first and the last residue of P. abyssi eS31 are not present in the structures. Cartoons were generated using UCSF Chimera (https://www.rbvi.ucsf.edu/chimera; accessed on 1 June 2024). Biomolecules 2024,14, 882 8 of 27 In consonance with the critical importance of yeast eS10, eS12 and eS31 r-proteins for cell growth, it has also been reported that loss-of-function mutations in the genes coding for these proteins in other model eukaryotes (Caenorhabditis elegans,Drosophila melanogaster, Danio rerio,Mus musculus,Homo sapiens) lead to a myriad of adverse phenotypes, including lethality, increased cell death, cell cycle arrest, reduced fertility, organ development defects and tumorigenesis [93]. 3. Roles of the Beak during Translation The head of the small r-subunit is a flexible and dynamic structure involved in the engagement of the mRNAs and tRNAs during translation. Taking into consideration the strategic position of the beak at the entrance of the mRNA channel in the small rsubunit, it is not surprising that the beak has been linked to diverse functions during the translation process: (i) During translation initiation, the beak is an important site for the interaction of trans-acting factors both in prokaryotic and eukaryotic ribosomes. For example, cryo-EM has revealed that the bacterial aldehyde-alcohol dehydrogenase E (AdhE) enzyme interacts with ribosomes in the beak region [ 94 ]. This enzyme provides a further RNA helicase activity, in addition to the intrinsic one of the ribosome [ 95 ], in order to ensure the linear configuration of structured mRNAs at the mRNA entrance to facilitate their translation [ 94 ]. In eukaryotes, several RNA helicases play roles during translation initiation. The canonical initiation factor eIF4A and the Ded1 (DDX3 in mammals) RNA helicase assist in the unwinding of the 5 ′ -UTR secondary structure of most mRNAs [ 96 – 98 ]. Interestingly, in mammals, the translation initiation of endogenous and viral mRNAs with highly structured 5 ′ -UTRs requires an additional RNA helicase, named DHX29 [ 99 , 100 ]. As revealed by cryo- EM, DHX29 contacts the beak and adjacent regions by interacting with at least uS3, eS10 and eS12 [ 101 ]. In other examples, the beak has been described as being important for the recognition of specific mRNAs. Accordingly, mammalian eS10 has been found to specifically interact with a class of cellular mRNAs containing the so-called TISU-element in their short 5′-UTRs [102]. (ii) The loading of the mRNA itself into the mRNA channel of the small r-subunit during translation initiation is regulated by the opening and closing of an mRNA latch situated below the beak that connects the body and the head of the small r-subunit [ 74 ]. The open conformation of this structure is promoted by the binding of distinct initiation factors (eIF1 and eIF1A in eukaryotes, IF1 in prokaryotes) to the small r-subunit during the formation of the eukaryotic 43S pre-initiation complex [ 103 ]. eIF1A is a globular protein harboring unstructured N- and C- terminal extensions of ca. 25 amino acids. During translation initiation, the globular domain of eIF1A is positioned at the A-site, while its extensions seem to project out of this site of the ribosome; thus, preventing tRNA binding to this site [ 103 ]. From X-ray crystallography, it can be inferred that the N-terminus of eIF1A directly contacts the eukaryote-specific N-terminal extension of eS31 and approaches extensions of other r-proteins, such as eS10, uS3 and uS19, that are adjacent to each other in the A-site [ 104 ]. These interactions seem to be crucial for translation as mutations in the N-terminal tail of eIF1A, which are frequently observed in several types of cancers [ 105 ], result in reduced binding of eIF1A to its r-protein partners and a hyperaccurate recognition of AUG codons that are embedded in an optimal sequence context [ 106 , 107 ]. Importantly, the phenotypic analysis of yeast ubi3 mutations (e.g., ubi3G75,76A) that interfere with the cleavage of the ubiquitin-eS31 fusion protein indicates that the non-cleaved protein can still assemble into mature 40S r-subunits, which are active in translation but mildly defective at the translation initiation stage. This defect is likely due to the interference of the ubiquitin moiety with the binding and proper activity of the initiator tRNA and the eIF1A factor [ 89 , 104 ]. Moreover, interactions between several subunits of the eIF3 complex and beak components, including eS10 (e.g., [ 108 ]), have been described to occur within the yeast 43S pre-initiation complex; these are expected to be of functional relevance during translation initiation (e.g., [109]). Biomolecules 2024,14, 882 9 of 27 (iii) The beak also participates in the formation of the binding surface for the internal ribosome entry site (IRES) of some viral mRNAs on human 40S r-subunits. For instance, among other 40S r-proteins, eS10 contributes to the binding of the hepatitis C virus (HCV) IRES [ 110 ]. Viral proteins also interact with the beak to hijack the host’s translation machinery. One interesting example concerns the SARS-CoV-2 coronavirus, whose non-structural protein NSP1 contains a globular N-terminal domain that binds the base of the beak, while its C-terminal extension blocks the mRNA channel entry site and thereby prevents any mRNA accommodation; thus, inhibiting translation of host mRNAs [ 111 – 113 ]. However, NSP1 does not impede the translation of viral mRNAs, which is promoted by the presence of a cis-acting RNA hairpin in the 5′-UTR of these mRNAs [114,115]. (iv) Translation is reversibly shutdown upon nutrient starvation in a variety of ways, including the accumulation of inactive or hibernating vacant 80S ribosomes. These dormant ribosomes contain eEF2 · GTP in the A-site and the hibernation factor Stm1 (in yeast) or SERBP1 (in mammals) in the mRNA channel, thereby impeding mRNA binding [ 87 , 116 ]. This mechanism of blocking the mRNA entry tunnel resembles that mentioned above for the coronavirus NSP1 protein. It has been described that the C-terminal region of Stm1/SERBP1 also stably associates with the head of 40S r-subunits, likely via binding to eS10, eS12 and eS31 [87,116]. (v) In all ribosomes, the interaction of the different translation elongation factors with the ribosome leads to specific movements of the head domain of the small r-subunit, including that of its associated beak towards the shoulder of the body of the same r-subunit (e.g., [ 117 , 118 ]). In eukaryotes, the beak components themselves interact with distinct domains of the translation elongation factors, as exemplified by the interaction of eS12 and eS31 with domains II and IV of eEF2 [ 7 ]. In agreement with the important role of beak r-proteins in the fidelity of translation elongation, the depletion of the essential yeast eS10 as well as the mutation or deletion of the genes encoding yeast eS12 and eS31 result in translation defects, including misreading [ 76 , 88 , 119 ]. Moreover, due to the specific position of the N-terminal extension of eS31 in the A-site of the ribosome, the assembly of non-cleaved yeast Ubi3 is expected to sterically interfere with the binding of the translation elongation factors to the ribosomal GTPase-associated center [89,91]. (vi) The beak is also expected to be functionally relevant during translation termination. Cryo-EM structures have revealed how eukaryotic translation termination factor 1 (eRF1), whose overall shape resembles a tRNA molecule, interacts with a stop codon in the A-site of the ribosome via its N-terminal lobe (e.g., [ 120 ] and references therein). Notably, in the structures of pre-termination complexes, a short segment of the N-terminal lobe of eRF1 is in close proximity to the initial residues of the N-terminal extension of eS31 [ 120 , 121 ]. Moreover, the mini-domain of eRF1, which is an insertion within the C-terminal domain, also interacts with the N-terminal extension of eS31 and protrudes toward the beak where it contacts helix h33 [120–122]. (vii) Another example of the role of the beak components in translation comes from studies of the cellular responses to elongation stalls induced by different stresses (including oxidative stress, heat shock or starvation) as well as by particular sequences, strong secondary structures and chemical damage within mRNAs. Normally, when exposed to stressful conditions, cells adapt by halting or decreasing the global synthesis of new proteins, while, concomitantly, inducing the selective translation of mRNAs encoding proteins that are necessary for cell survival and stress recovery [ 123 ]. This translational reprogramming can be mediated by multiple parallel and independent signaling pathways that converge on the modulation of the function of a few key translation factors [ 124 ]. Relatively recent studies from the Silva laboratory showed that following oxidative stress, induced by an exposure of yeast cells to hydrogen peroxide, a set of r-proteins were K63-specifically polyubiquitinated at different residues by the ubiquitin-conjugating enzyme Rad6 and the ubiquitin-protein ligase Bre1, with the extent of this modification declining very rapidly during stress recovery [ 125 , 126 ]. Most of these r-proteins are located within the head of the small r-subunit of the ribosome and include uS3, the beak components eS10, eS12 and eS31 Biomolecules 2024,14, 882 16 of 27 Ribosomopathies are a group of rare inherited or acquired genetic diseases linked to defects in r-proteins or ribosome biogenesis factors [ 207 – 209 ]. Despite the importance of ribosomes in all cell types, these diseases result mainly in tissue-specific manifestations, especially in the hematopoietic system [ 207 ]. Intriguingly, inherited ribosomopathies are congenital and normally exhibit a paradoxical transition from early symptoms related to cellular hypo-proliferation to a hyper-proliferative oncogenic state later in life [ 210 ]. The best studied ribosomopathy, which is also one of the most prevalent ones (10 individuals per million live births) is the so-called Diamond–Blackfan Anemia (DBA) [ 211 ]. DBA is mostly a dominant genetic disorder (autosomal or X-linked) that is characterized by the reduced formation of red blood cells and is also associated with a series of other congenital anomalies, such as skeletal abnormalities, heart and genitourinary malformations, and an increased cancer susceptibility [ 212 ]. Most patients diagnosed with DBA harbor heterozygous loss-of-function mutations in particular genes encoding r-proteins, either of the small or the large r-subunit [ 212 ]. About 3% of all DBA patients have been reported to carry mutations in the RPS10 gene, most likely leading in all cases to the production of non-functional eS10 variant proteins [ 212 , 213 ]; these mutations mostly consist of (i) insertion mutations that cause a frameshift and the appearance of a premature termination codon, (ii) nonsense mutations, namely, changes of particular codons to a stop codon (often, the R113Stop mutation), (iii) missense mutations that transform the RPS10 start codon into an isoleucine or a threonine codon (M1I or M1T), and (iv) different other missense mutations (e.g., L14F, P30L) of so far unknown biological significance ([ 213 , 214 ]; for more details, check the UniProt entry P46783 and the OMIM entry 603632). Mutations in the human RPS27A gene, which codes for human eS31, have also been identified in patients with DBA (e.g., S57P); however, whether these mutations are indeed pathogenic genetic variants remains to be determined [ 215 ]. To our knowledge, no DBA-linked mutations have so far been reported in the RPS12 gene. However, as the underlying mutations in at least 20% of patients with DBA syndromes have not yet been identified [ 212 ], it is still possible that RPS12 alleles could be responsible for DBA manifestations; in line with this possibility, RPS12 haploinsufficiency in mice leads to an erythropoiesis defect that recapitulates the one found in DBA patients (discussed in [ 216 ]). From a very simplistic point of view, as the DBA disease is mostly caused by loss-of-function mutations in several r-protein and a few RAF genes, all associated DBA symptoms and manifestations must come from common dysfunctions of the same molecular process that, in this case, can be no other than ribosome biogenesis [ 217 ], ultimately leading to an impairment or a limitation of translation. Thus, in a non-exclusive manner, it has been proposed that (i) the hypo-proliferative, pro-apoptotic anemia associated with DBA could be the consequence of a global reduction in translation, limiting below a critical threshold the synthesis of critical proteins, such as the globins and the transcription factor GATA1, with the latter being essential for normal erythropoiesis [ 218 ]. In agreement with this possibility, GATA1 translation is reduced in erythroid precursor cells of DBA patients with mutations in different r-protein genes (e.g., [ 219 – 221 ]), and loss-of-function mutations in GATA1 result in a DBA-like phenotype (e.g., [ 222 , 223 ] and references therein). (ii) DBA cells display elevated levels of reactive oxygen species (ROS), which, by generating a high oxidative stress, inhibit cell proliferation [ 206 , 207 ]. In this regard, lowering cellular ROS levels by antioxidants can rescue the proliferation defects in cells subjected to r-protein haploinsufficiency or carrying selected r-protein mutations [ 207 , 224 ]. (iii) As a consequence of the ribosome biogenesis deficiency occurring in DBA cells, the so-called nucleolar stress response is triggered in these cells, which induces p53 stabilization and enables p53 to transactivate its target genes, leading to cell cycle arrest, apoptosis, autophagy, and senescence [ 225 , 226 ]. In normal growth conditions, cellular levels of p53 are maintained low due to its efficient recognition and ubiquitination by the E3 ubiquitin ligase MDM2 (HDM2 in humans) and the subsequent degradation of ubiquitinated p53 by the proteasome. However, when ribosome biogenesis is impaired, non-assembled r-proteins tend to accumulate and can be released from the nucleolus to the nucleoplasm. Several different free r-proteins, but Biomolecules 2024,14, 882 17 of 27 primarily uL5 and uL18 as part of the 5S RNP, can bind and sequester MDM2, thereby preventing the degradation of p53; thus, the upregulation of p53 explains many of the hypo-proliferative phenotypes displayed by DBA patients, including bone marrow erythroid hypoplasia [ 206 , 227 , 228 ]. In consonance with the relevant role of nucleolar stress in DBA, genetic or pharmacological inactivation of p53 can rescue disease-associated phenotypes [ 206 , 209 , 225 , 229 ]. Interestingly, it has been shown that eS31 is able to regulate the MDM2-p53 loop in response to nucleolar stress [ 230 , 231 ]. Moreover, eS31 apparently interacts with the central acidic domain of MDM2 through its eukaryote-specific N-terminal extension [ 230 ]. Therefore, this interaction seems not to be mutually exclusive from the ones of MDM2 with p53, which used a short, N-terminal segment to bind to the N-terminal domain of MDM2 [ 232 ], and with the r-proteins uL5 and uL18, which mostly involve the Zn-finger and RING domains of MDM2 [ 59 , 233 ]. Importantly, the overexpression of eS31 reduces MDM2-mediated ubiquitination of p53, thereby leading to its stabilization and activation [ 230 , 234 ]. The induction of p53 by eS31 may likely be additionally fueled by the observation that the RPS27A gene is apparently also transcriptionally activated by p53 [ 234 ]. Moreover, it has also been shown that MDM2 mediates the ubiquitination and proteasomal degradation of eS31 in response to nucleolar stress, indicating that free eS31 could be a physiological substrate of MDM2 [ 230 ]. It has been proposed that this mutual inhibitory regulation between MDM2 and eS31 may contribute to cellular recovery after the experienced stress [ 230 ]. Another report has suggested that the RAF PICT1 seems to regulate the interaction between eS31 and MDM2, as low levels of PICT1 are apparently required for the efficient translocation of eS31 from the nucleolus to the nucleoplasm, so that it can bind MDM2 [ 235 ]. It has also been described in several human lung cancer cell lines that eS31 could interact with uL5 in a way that might weaken the strength of the interaction between uL5 and MDM2; thus, knockdown of RPS27A stabilizes p53 in a uL5-dependent manner, promoting the p53 tumor suppressor functions [ 231 ]. Altogether, the above-mentioned data highlight that eS31 is connected to the MDM2-p53 axis and suggest that eS31 may be relevant for fine-tuning the cellular response to and recovery from nucleolar stress. However, its importance is apparently cell-type dependent, as recently shown by the Schneider group, who demonstrated that knockdown of RPS27A robustly induced p53 in certain cell lines but not in others [181]. Cancer cells require a high production of ribosomes to sustain boundless growth and cell division (e.g., [ 236 ]). Moreover, many r-proteins, including the beak ones, have been implicated in cancer development (e.g., [ 206 , 237 – 239 ]). Mutations and altered expression of beak r-proteins have been described in many cancer types, in some cases likely displaying an extra-ribosomal function: (i) high expression of RPS10 has been found in colorectal, renal and prostate cancer [ 240 ], whereas it has been reported that ribosomes purified from MDA-MB-231 breast cancer cells contain substoichiometric levels of eS10, among other r-proteins [ 119 ]. (ii) Overexpression of RPS12 has been observed in colon adenomatous polyps and carcinomas as well as in gastric cancers [ 241 ]. Deletions of RPS12 are frequently observed in diffuse large B cell lymphomas [ 242 ], and eS12 has been reported to play a role as a stimulator of WNT secretion in cancer cells, which is particularly important in the context of triple-negative breast cancer initiation and progression [ 243 ]. (iii) It has been reported that eS31 is overexpressed in renal, colon, cervical, and breast cancers, chronic myeloid leukemia and lung adenocarcinoma [ 161 , 231 , 244 , 245 ]. In most of these cases, the overexpression of eS31 correlates with poor prognosis for the patients. A recent review has highlighted the expression and role of eS31 in cancer cells and tumor tissues [161]. 7. Concluding Remarks and Future Perspectives Herein, we have discussed the relevance of the beak, a structurally conserved region of the small r-subunit of cytoplasmic ribosomes in all three domains of life. Notably, the beak has transitioned from a structure composed exclusively of rRNA in bacteria to a protuberance comprising three specific r-proteins, eS10, eS12 and eS31, in eukaryotes, while an intermediate situation prevails in archaea as the beaks of many species only contain two Biomolecules 2024,14, 882 18 of 27 r-proteins, eS31 and eL8, with the latter being clearly the ancestor of the eukaryotic eS12 r-protein. As also discussed, the beak has diverged considerably in the mitoribosomes of some organisms, such as in trypanosomatids, perhaps owing to the particular translation requirements inside these organelles [ 246 , 247 ]. As outlined in this review, the beak has important roles in the three major phases of translation (initiation, elongation and termination) and is involved in many other events of the translation process, including ribosome stalling and collisions, as well as other seemingly translation-unrelated processes, such as cell competition in Drosophila. In many of these processes, the biological significance of post-translational modifications, such as ubiquitination, is still poorly understood. We have also highlighted the implication of the beak r-proteins in the stepwise assembly of nascent 40S r-subunits. Regarding the maturation of the eukaryotic beak, it is evident that further research is required to precisely define the assembly timing of the beak r-proteins; for instance, it is still controversial whether eS31 associates with 40S r-subunit maturation intermediates in the nucleus or the cytoplasm. More studies are also required to elucidate whether the beak r-proteins play active or passive roles during the assembly and nuclear export of pre-40S r-subunits. Finally, we discussed the relevance of the beak r-proteins in human diseases, especially ribosomopathies and cancer. A deeper understanding of the connection between these diseases and the ribosome biogenesis process is expected to offer new perspectives for therapeutic approaches. Author Contributions: S.M.-V., C.V.G., C.R.-H., D.K. and J.d.l.C. wrote the manuscript. D.K. and J.d.l.C. edited the draft and the final version of the manuscript. All authors have read and agreed to the published version of the manuscript. Funding: This publication is part of the project R+D+i PID2022-136564NB-I00 funded by MCIN/AEI/10.13039/501100011033/ERDF, EU to J.d.l.C. We also acknowledge the Andalusian Platform for Biomodels and Resources in Genomic Edition, the Fortalece Program (FORT 2023) from MCIN, and the Translacore (CA21154) and ProteoCure (CA20113) COST Actions from the EU for support. D. K. is supported by the Swiss National Science Foundation (project grant 310030_204801). S.M.-V. was an academic research staff member of the Andalusian Research, Development, and Innovation Plan by the Andalusian Regional Government (PAIDI 2020). C.V.G. was supported by a Marie Sklodowska-Curie Individual Fellowship (H2020-MSCA-IF-2020, Grant 101024158 from the EU). C.R.-H. is supported by a María Zambrano Grant from the Spanish Ministry of Universities. Acknowledgments: We thank members of the de la Cruz laboratory for valuable discussions, critical reading, and suggestions. Conflicts of Interest: The authors declare no conflicts of interest. References 1. Wilson, D.N.; Doudna Cate, J.H. The structure and function of the eukaryotic ribosome. Cold Spring Harb. Perspect. Biol. 2012, 4, a011536. [CrossRef] [PubMed] 2. Melnikov, S.; Ben-Shem, A.; Garreau de Loubresse, N.; Jenner, L.; Yusupova, G.; Yusupov, M. One core, two shells: Bacterial and eukaryotic ribosomes. Nat. Struct. Mol. Biol. 2012,19, 560–567. [CrossRef] [PubMed] 3. Greber, B.J.; Ban, N. 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