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Protein synthesis is a cellular process essential for life. Ribosomes are responsible for protein synthesis in all cells. Ribosomes are composed of three RNA molecules (23S, 16S, and 5S ribosomal RNAs) and about 50 proteins. The ribosomal RNAs play a central role in protein synthesis by forming the active site for peptide bond formation and by interacting with messenger RNA, transfer RNA, and translational factors. Here we describe the functional role of the ribosomal RNAs in protein synthesis.

Nascent ribosomal 60S subunits undergo the last maturation steps in the cytoplasm. The last one involves removing the anti-association factor eIF6 from the 60S ribosomal surface by the joint action of the Elongation Factor-like 1 (EFL1) GTPase and the SBDS protein. Herein, we studied the evolutionary relationship of the EFL1 and EF-2 protein families and the functional conservation within EFL1 orthologues. Phylogenetic analysis demonstrated that the EFL1 proteins are exclusive of eukaryotes and share an evolutionary origin with the EF-2 and EF-G protein families. EFL1 proteins originated by gene duplication from the EF-2 proteins and specialized in ribosome maturation while the latter retained their function in translation. Some organisms have more than one EFL1 protein resulting from alternative splicing, while others are encoded in different genes originated by gene duplication. However, the function of these alternative EFL1 proteins is still unknown. We performed GTPase activity and complementation assays to study the functional conservation of EFL1 homologs alone and together with their SBDS counterparts. None of the orthologues or cross-species combinations could replace the function of the corresponding yeast EFL1•SBDS binomial. The complementation of SBDS interspecies chimeras indicates that domain 2 is vital for its function together with EFL1 and the 60S subunit. The results suggest a functional species-specificity and possible co-evolution between EFL1, SBDS, and the 60S ribosomal subunit. These findings set the basis for further studies directed to understand the molecular evolution of these proteins and their impact on ribosome biogenesis and disease.

BCCIP was isolated based on its interactions with tumor suppressors BRCA2 and p21. Knockdown or knockout of BCCIP causes embryonic lethality in mice. BCCIP deficient cells exhibit impaired cell proliferation and chromosome instability. BCCIP also plays a key role in biogenesis of ribosome 60S subunits. BCCIP is conserved from yeast to humans, but it has no discernible sequence similarity to proteins of known structures. Here we report two crystal structures of an N-terminal truncated human BCCIPβ, consisting of residues 61-314. Structurally BCCIP is similar to GCN5-related acetyltransferases (GNATs) but contains different sequence motifs. Moreover, both acetyl-CoA and substrate-binding grooves are altered in BCCIP. A large 19-residue flap over the putative CoA binding site adopts either an open or closed conformation in BCCIP. The substrate binding groove is significantly reduced in size and is positively charged despite the acidic isoelectric point of BCCIP. BCCIP has potential binding sites for partner proteins and may have enzymatic activity.

The nascent polypeptide exit tunnel (NPET) is a major functional center of 60S ribosomal subunits. However, little is known about how the NPET is constructed during ribosome assembly. We utilized molecular genetics, biochemistry, and cryo-electron microscopy (cryo-EM) to investigate the functions of two NPET-associated proteins, ribosomal protein uL4 and assembly factor Nog1, in NPET assembly. Structures of mutant pre-ribosomes lacking the tunnel domain of uL4 reveal a misassembled NPET, including an aberrantly flexible ribosomal RNA helix 74, resulting in at least three different blocks in 60S assembly. Structures of pre-ribosomes lacking the C-terminal extension of Nog1 demonstrate that this extension scaffolds the tunnel domain of uL4 in the NPET to help maintain stability in the core of pre-60S subunits. Our data reveal that uL4 and Nog1 work together in the maturation of ribosomal RNA helix 74, which is required to ensure proper construction of the NPET and 60S ribosomal subunits., The nascent polypeptide exit tunnel (NPET) is a functional center of the large ribosomal subunit through which the nascent polypeptide chains travel from the peptidyltransferase center (PTC). Here the authors provide structural insight into NPET maturation and how it is linked to other aspects of ribosome biogenesis.

The balance of ribosomal proteins is important for the assembly of ribosomal subunits and cell viability. The synthesis of ribosomal proteins in a eukaryotic cell is controlled by various mechanisms, including autoregulation, which so far has been revealed for only a few of these proteins. We applied the photoactivatable 4-thiouridine-enhanced cross-linking and immunoprecipitation assay to HEK293T cells overproducing FLAG-labeled human ribosomal protein eL29 (eL29FLAG) to determine which RNAs other than rRNA interact with eL29. We demonstrated that eL29FLAG was incorporated into 60S subunits, and that ribosomes with those containing eL29FLAG were competent in translation. Analysis of the next generation sequencing data obtained from a DNA library derived from RNA fragments with covalently attached eL29FLAG peptide residues showed that the protein was cross-linked to the mRNA of the eL29-coding gene, which turned out to be its only major RNA target. The eL29FLAG cross-linking sites were located in the 3′ part of the mRNA coding sequence (CDS). A specific helix that mimics the eL29 binding site on 28S rRNA was proposed as a site that is recognized by the protein upon its binding to the cognate mRNA. In addition, it was found that both eL29FLAG mRNA and eL29 mRNA, unlike those of other ribosomal proteins, were co-immunoprecipitated with eL29FLAG from the ribosome-depleted cell lysate, and recombinant eL29 inhibited the translation of the eL29 mRNA CDS transcript in a cell-free system. All this suggests that human eL29 regulates its own synthesis via a feedback mechanism by binding to the cognate mRNA, preventing its translation.

Ribosome biogenesis is crucial for cellular metabolism and has important implications for disease and aging. Human (Homo sapiens) glioma tumor-suppressor candidate region gene2 (GLTSCR2) and yeast (Saccharomyces cerevisiae) Nucleolar protein53 (Nop53) are orthologous proteins with demonstrated roles as ribosome biogenesis factors; knockdown of GLTSCR2 impairs maturation of 18S and 5.8S ribosomal RNAs (rRNAs), and Nop53 is required for maturation of 5.8S and 25S rRNAs. Here, we characterized SMALL ORGAN4 (SMO4), the most likely ortholog of human GLTSCR2 and yeast Nop53 in Arabidopsis (Arabidopsis thaliana). Loss of function of SMO4 results in a mild morphological phenotype; however, we found that smo4 mutants exhibit strong cytological and molecular phenotypes: nucleolar hypertrophy and disorganization, overaccumulation of 5.8S and 18S rRNA precursors, and an imbalanced 40S:60S ribosome subunit ratio. Like yeast Nop53 and human GLTSCR2, Arabidopsis SMO4 participates in 5.8S rRNA maturation. In yeast, Nop53 cooperates with mRNA transport4 (Mtr4) for 5.8S rRNA maturation. In Arabidopsis, we found that SMO4 plays similar roles in the 5.8S rRNA maturation pathway than those described for MTR4. However, SMO4 seems not to participate in the degradation of by-products derived from the 5'-external transcribed spacer (ETS) of 45S pre-rRNA, as MTR4 does.

Cytosolic 80S ribosomes contain proteins of the mature cytosolic ribosome (r-proteins) as well as proteins with roles in ribosome biogenesis, protein folding or modification. Here, we refined the core r-protein composition in Arabidopsis thaliana by determining the abundance of different proteins during enrichment of ribosomes from cell cultures using peptide mass spectrometry. The turnover rates of 26 40S subunit r-proteins and 29 60S subunit r-proteins were also determined, showing that half of the ribosome population is replaced every 3–4 days. Three enriched proteins showed significantly shorter half-lives; a protein annotated as a ribosomal protein uL10 (RPP0D, At1g25260) with a half-life of 0.5 days and RACK1b and c with half-lives of 1–1.4 days. The At1g25260 protein is a homologue of the human Mrt4 protein, a trans-acting factor in the assembly of the pre-60S particle, while RACK1 has known regulatory roles in cell function beyond its role in the 40S subunit. Our experiments also identified 58 proteins that are not from r-protein families but co-purify with ribosomes and co-express with r-proteins; 26 were enriched more than 10-fold during ribosome enrichment. Some of these enriched proteins have known roles in translation, while others are newly proposed ribosome-associated factors in plants. This analysis provides an improved understanding of A. thaliana ribosome protein content, shows that most r-proteins turnover in unison in vivo, identifies a novel set of potential plant translatome components, and how protein turnover can help identify r-proteins involved in ribosome biogenesis or regulation in plants.

The protein composition and structure of assembling 60S ribosomal subunits undergo numerous changes as pre-ribosomes transition from the nucleolus to the nucleoplasm. This includes stable anchoring of the Rpf2 subcomplex containing 5S rRNA, rpL5, rpL11, Rpf2 and Rrs1, which initially docks onto the flexible domain V of rRNA at earlier stages of assembly. In this work, we tested the function of the C-terminal domain (CTD) of Rpf2 during these anchoring steps, by truncating this extension and assaying effects on middle stages of subunit maturation. The rpf2Δ255-344 mutation affects proper folding of rRNA helices H68-70 during anchoring of the Rpf2 subcomplex. In addition, several assembly factors (AFs) are absent from pre-ribosomes or in altered conformations. Consequently, major remodeling events fail to occur: rotation of the 5S RNP, maturation of the peptidyl transferase center (PTC) and the nascent polypeptide exit tunnel (NPET), and export of assembling subunits to the cytoplasm., As assembling 60S subunits transit from the nucleolus to the nucleoplasm, they undergo significant changes in protein composition and structure. Here, the authors provide a structural view of interconnected events during the middle steps of assembly that include the maturation of the central protuberance, the peptidyltransferase center and the nascent polypeptide exit tunnel.

Indirect somatic genetic rescue (SGR) of a germline mutation is thought to be rare in inherited Mendelian disorders. Here, we establish that acquired mutations in the EIF6 gene are a frequent mechanism of SGR in Shwachman-Diamond syndrome (SDS), a leukemia predisposition disorder caused by a germline defect in ribosome assembly. Biallelic mutations in the SBDS or EFL1 genes in SDS impair release of the anti-association factor eIF6 from the 60S ribosomal subunit, a key step in the translational activation of ribosomes. Here, we identify diverse mosaic somatic genetic events (point mutations, interstitial deletion, reciprocal chromosomal translocation) in SDS hematopoietic cells that reduce eIF6 expression or disrupt its interaction with the 60S subunit, thereby conferring a selective advantage over non-modified cells. SDS-related somatic EIF6 missense mutations that reduce eIF6 dosage or eIF6 binding to the 60S subunit suppress the defects in ribosome assembly and protein synthesis across multiple SBDS-deficient species including yeast, Dictyostelium and Drosophila. Our data suggest that SGR is a universal phenomenon that may influence the clinical evolution of diverse Mendelian disorders and support eIF6 suppressor mimics as a therapeutic strategy in SDS., Shwachman-Diamond syndrome (SDS) is a leukemia predisposition disorder that is caused by defective release of eIF6 during ribosome assembly. Here the authors show that acquired somatic EIF6 mutations are frequent in the hematopoietic cells from individuals with SDS and provide a selective advantage over non-modified cells.

In actively translating 80S ribosomes the ribosomal protein eS7 of the 40S subunit is monoubiquitinated by the E3 ligase Not41,2 and deubiquitinated by the deubiquitination enzyme Otu2 upon ribosomal subunit recycling3. Despite its importance for general efficiency of translation the exact role and structural basis for this specific translational reset are only poorly understood. Here we present biochemical and structural data showing that Otu2 can engage the recycled 40S subunit together with the recycling factors ABCE1 and Tma64 immediately after 60S dissociation for mRNA recycling, and that it dissociates before 48S initiation complex formation. A combined structural analysis of Otu2 and Otu2-40S complexes by X-ray crystallography, AlphaFold2 prediction4 and cryo-EM revealed how Otu2 can specifically be recruited to the 40S, but not to the 80S ribosome, for removal of the eS7-bound ubiquitin moiety. Here, interactions of the largely helical N-terminal domain of Otu2 to sites that are masked and therefore inaccessible in the 80S ribosome are of crucial importance. Collectively, we provide the structural basis for the Otu2 driven deubiquitination step providing a first mechanistic understanding of this translational reset step during ribosome recycling/(re)initiation.

Ribosomal biogenesis has been studied by biochemical, genetic and electron microscopic approaches, but live cell data on the in vivo kinetics are still missing. Here we analyse the export kinetics of the large ribosomal subunit (pre-60S particle) through single NPCs in human cells. We established a stable cell line co-expressing Halo-tagged eIF6 and GFP-fused NTF2 to simultaneously label pre-60S particles and NPCs, respectively. By combining single molecule tracking and super resolution confocal microscopy we visualize the dynamics of single pre-60S particles during export through single NPCs. For export events, maximum particle accumulation is found in the centre of the pore, while unsuccessful export terminates within the nuclear basket. The export has a single rate limiting step and a duration of ∼24 milliseconds. Only about 1/3 of attempted export events are successful. Our results show that the mass flux through a single NPC can reach up to ~125 MDa·s−1 in vivo., Nature Communications, 12 (1), ISSN:2041-1723

Synthesis of eukaryotic ribosomes involves the assembly and maturation of precursor particles (pre-ribosomal particles) containing ribosomal RNA (rRNA) precursors, ribosomal proteins (RPs) and a plethora of assembly factors (AFs). Formation of the earliest precursors of the 60S ribosomal subunit (pre-60S r-particle) is among the least understood stages of ribosome biogenesis. It involves the Npa1 complex, a protein module suggested to play a key role in the early structuring of the pre-rRNA. Npa1 displays genetic interactions with the DExD-box protein Dbp7 and interacts physically with the snR190 box C/D snoRNA. We show here that snR190 functions as a snoRNA chaperone, which likely cooperates with the Npa1 complex to initiate compaction of the pre-rRNA in early pre-60S r-particles. We further show that Dbp7 regulates the dynamic base-pairing between snR190 and the pre-rRNA within the earliest pre-60S r-particles, thereby participating in structuring the peptidyl transferase center (PTC) of the large ribosomal subunit., The Henry/Henras group is supported by grants from ANR (ANR-20-CE12-0026) and funding from CNRS and University of Toulouse. R.A.M. is supported by grants from the Rectorat of Lebanese University. M.J. is supported by a Ph.D. fellowship from the Lebanese University and CIOES Organization. The group of J.d.l.C. is supported by the Spanish Ministry of Science and Innovation [PID2019-103859-GB-I00 AEI/ 10.13039/501100011033], and the Andalusian Regional Government (JA; BIO-271). J.C. was supported by a Ph.D. fellowship (PIF) from the University of Seville, and S.M.-V. is an academic research staff of the JA (PAIDI2020). M.T.B. and K.E.B. are supported by funding from the Deutsche Forschungsgemeinschaft (SFB860) and the University Medical Centre Göttingen.

Many unannotated microproteins and alternative proteins (alt-proteins) have recently been found to be co-encoded with canonical proteins, but few of their functions are known. Motivated by the hypothesis that alt-proteins undergoing active or stress-induced synthesis could play important cellular roles, here, we developed a chemoproteomic pipeline to identify nascent alt-proteins in human cells. We identified 22 actively translated unannotated alt-proteins, one of which is upregulated after DNA damage stress. We further defined MINAS-60 (MIcroprotein thatNegatively regulatesASsembly of the pre-60S ribosomal subunit), a nucleolar localized alt-protein co-encoded with human RBM10.Depletion of MINAS-60 increases the amount of the mature 60S ribosomal subunit, consequently upregulating global protein synthesis and cell proliferation by repressing late-stage pre-60S assembly and export of the 60S ribosome subunit to the cytoplasm. Together, these results implicate MINAS-60 as a repressor of ribosome biogenesis, and demonstrate that chemoproteomics can enable generation of functional hypotheses for uncharacterized alt-proteins.

Protein synthesis is a cyclical process consisting of translation initiation, elongation, termination and ribosome recycling. The release factors SBDS and EFL1 (both mutated in the leukaemia predisposition disorder Shwachman-Diamond syndrome) license entry of nascent 60S ribosomal subunits into active translation by evicting the anti-association factor eIF6 from the 60S intersubunit face. Here, we show that in mammalian cells, eIF6 holds all free cytoplasmic 60S subunits in a translationally inactive state and that SBDS and EFL1 are the minimal components required to recycle these 60S subunits back into additional rounds of translation by evicting eIF6. Increasing the dose of eIF6 in mice in vivo impairs terminal erythropoiesis by sequestering post-termination 60S subunits in the cytoplasm, disrupting subunit joining and attenuating global protein synthesis. Our data reveal that ribosome maturation and recycling are dynamically coupled by a mechanism that is disrupted in an inherited leukaemia predisposition disorder.

The decay of messenger RNA with a premature termination codon (PTC) by nonsense mediated decay (NMD) is an important regulatory pathway for eukaryotes and an essential pathway in mammals. NMD is typically triggered by the ribosome terminating at a stop codon that is aberrantly distant from the poly-A tail. Here, we use a fluorescence screen to identify factors involved in NMD in S. cerevisiae. In addition to the known NMD factors, including the entire UPF family (UPF1, UPF2 and UPF3), as well as NMD4 and EBS1, we identify factors known to function in post-termination recycling and characterize their contribution to NMD. We then use a series of modified reporter constructs that block both elongating and scanning ribosomes downstream of stop codons and demonstrate that a deficiency in recycling of 80S ribosomes or 40S subunits stabilizes NMD substrates. These observations in S. cerevisiae expand on recently reported data in mammals indicating that the 60S recycling factor ABCE1 is important for NMD (1, 2) by showing that increased activities of both elongating and scanning ribosomes (80S or 40S) in the 3’UTR correlate with a loss of NMD.Author SummaryIn this work, we aim to understand the mechanism of targeting mRNAs for decay via the long-studied nonsense mediated decay (NMD) pathway. We demonstrate that efficient large and small subunit ribosome recycling are necessary components of NMD. We go on to provide evidence that either scanning or actively translating ribosomes in the 3’UTR disrupt the decay of NMD targets. Our work highlights the importance of the composition of the 3’UTR in NMD signaling and emphasizes the need for this region to indeed be untranslated for NMD to occur. Exon junction complexes (EJCs) in the 3’UTR are known to induce NMD, however, in the budding yeast system used here, the NMD targets are EJC-free. Therefore, our data support a model in which factors other than EJCs may accumulate in the 3’UTR and provide a signal for NMD.

Eukaryotic ribosomal biogenesis is a high-energy–demanding and complex process that requires hundreds of trans-acting factors to dynamically build the highly-organized 40S and 60S subunits. Each ribonucleoprotein complex comprises specific rRNAs and ribosomal proteins that are organized into functional domains. The RNA exosome complex plays a crucial role as one of the pre-60S–processing factors, because it is the RNase responsible for processing the 7S pre-rRNA to the mature 5.8S rRNA. The yeast pre-60S assembly factor Nop53 has previously been shown to associate with the nucleoplasmic pre-60S in a region containing the “foot” structure assembled around the 3′ end of the 7S pre-rRNA. Nop53 interacts with 25S rRNA and with several 60S assembly factors, including the RNA exosome, specifically, with its catalytic subunit Rrp6 and with the exosome-associated RNA helicase Mtr4. Nop53 is therefore considered the adaptor responsible for recruiting the exosome complex for 7S processing. Here, using proteomics-based approaches in budding yeast to analyze the effects of Nop53 on the exosome interactome, we found that the exosome binds pre-ribosomal complexes early during the ribosome maturation pathway. We also identified interactions through which Nop53 modulates exosome activity in the context of 60S maturation and provide evidence that in addition to recruiting the exosome, Nop53 may also be important for positioning the exosome during 7S processing. On the basis of these findings, we propose that the exosome is recruited much earlier during ribosome assembly than previously thought, suggesting the existence of additional interactions that remain to be described.

Aberrantly stalled ribosomes initiate the ribosome-associated quality control (RQC) and mRNA surveillance pathways for the degradation of potentially toxic peptides and faulty mRNAs. During RQC, ANKZF1 (yeast Vms1p) releases ubiquitinated nascent proteins from 60S ribosomal subunits for proteasomal degradation. Here, we use a cell-free system to show that ANKZF1 and Vms1p sever polypeptidyl-tRNAs on RQC complexes by precisely cleaving off the terminal 3'CCA nucleotides universal to all tRNAs. This produces a tRNA fragment that cannot be aminoacylated until its 3'CCA end is restored. The recycling of ANKZF1-cleaved tRNAs is intact in the mammalian cytosol via a two-step process that requires the removal of a 2',3'-cyclic phosphate and TRNT1, the sole CCA-adding enzyme that mediates tRNA biogenesis in eukaryotes. TRNT1 also discriminates between properly folded tRNA substrates and aberrant tRNA substrates, selectively tagging the latter for degradation. Thus, ANKZF1 liberates peptidyl-tRNAs from stalled ribosomes such that the tRNA is checked in an obligate way for integrity before reentry into the translation cycle.

One of the final maturation steps of the large ribosomal subunit requires the joint action of the elongation factor-like 1 (human EFL1, yeast Efl1) GTPase and the Shwachman-Diamond syndrome protein (human SBDS, yeast Sdo1) to release the eukaryotic translation initiation factor 6 (human eIF6, yeast Tif6) and allow the assembly of mature ribosomes. EFL1 function is driven by conformational changes. However, the nature of such conformational changes or the mechanism by which they are prompted are still largely unknown. In previous studies, it has been established that this GTPase interacts with its cofactor in solution in an inverted orientation with respect to the binding mode derived from 60S ribosome subunit cryo-EM data. To shed new light on this conundrum, we characterized calorimetrically the energetic basis describing the recognition of Efl1 to GT(D)P, Sdo1 and their intercommunication in solution. A structural-based analysis of the binding signatures indicates that Efl1 has a large structural flexibility. The mutual effects of Sdo1 and nucleotides on Efl1 modulate in a very specific and robust way the complex conformational landscape of Efl1, resembling the behavior observed with other GTPases and their cofactors.

Summary: 18S non-functional rRNA decay (NRD) eliminates non-functional 18S rRNA with deleterious mutations in the decoding center. Dissociation of the non-functional 80S ribosome into 40S and 60S subunits is a prerequisite step for degradation of the non-functional 18S rRNA. However, the mechanisms by which the non-functional ribosome is recognized and dissociated into subunits remain elusive. Here, we report that the sequential ubiquitination of non-functional ribosomes is crucial for subunit dissociation. 18S NRD requires Mag2-mediated monoubiquitination followed by Hel2- and Rsp5-mediated K63-linked polyubiquitination of uS3 at the 212th lysine residue. Determination of the aberrant 18S rRNA levels in sucrose gradient fractions revealed that the subunit dissociation of stalled ribosomes requires sequential ubiquitination of uS3 by E3 ligases and ATPase activity of Slh1 (Rqt2), as well as Asc1 and Dom34. We propose that sequential uS3 ubiquitination of the non-functional 80S ribosome induces subunit dissociation by Slh1, leading to degradation of the non-functional 18S rRNA. : 18S non-functional rRNA decay (NRD) eliminates non-functional 18S rRNA with deleterious mutations in the decoding center. Sugiyama et al. show that 18S NRD requires Mag2-mediated monoubiquitination followed by Hel2- and Rsp5-mediated K63-linked polyubiquitination of uS3. Subunit dissociation requires Asc1, Dom34, sequential ubiquitination of uS3, and ATPase activity of Slh1 (Rqt2). Keywords: non-functional rRNA decay, ribosome ubiquitination, sequential ubiquitination, RQT complex, subunit dissociation

Ribosome biogenesis is essential for plants to successfully acclimate to low temperature. Without dedicated steps supervising the 60S large subunits (LSUs) maturation in the cytosol, e.g., Rei-like (REIL) factors, plants fail to accumulate dry weight and fail to grow at suboptimal low temperatures. Around REIL, the final 60S cytosolic maturation steps include proofreading and assembly of functional ribosomal centers such as the polypeptide exit tunnel and the P-Stalk, respectively. In consequence, these ribosomal substructures and their assembly, especially during low temperatures, might be changed and provoke the need for dedicated quality controls. To test this, we blocked ribosome maturation during cold acclimation using two independent reil double mutant genotypes and tested changes in their ribosomal proteomes. Additionally, we normalized our mutant datasets using as a blank the cold responsiveness of a wild-type Arabidopsis genotype. This allowed us to neglect any reil-specific effects that may happen due to the presence or absence of the factor during LSU cytosolic maturation, thus allowing us to test for cold-induced changes that happen in the early nucleolar biogenesis. As a result, we report that cold acclimation triggers a reprogramming in the structural ribosomal proteome. The reprogramming alters the abundance of specific RP families and/or paralogs in non-translational LSU and translational polysome fractions, a phenomenon known as substoichiometry. Next, we tested whether the cold-substoichiometry was spatially confined to specific regions of the complex. In terms of RP proteoforms, we report that remodeling of ribosomes after a cold stimulus is significantly constrained to the polypeptide exit tunnel (PET), i.e., REIL factor binding and functional site. In terms of RP transcripts, cold acclimation induces changes in RP families or paralogs that are significantly constrained to the P-Stalk and the ribosomal head. The three modulated substructures represent possible targets of mechanisms that may constrain translation by controlled ribosome heterogeneity. We propose that non-random ribosome heterogeneity controlled by specialized biogenesis mechanisms may contribute to a preferential or ultimately even rigorous selection of transcripts needed for rapid proteome shifts and successful acclimation.

The synthesis of new proteins is a fundamental aspect of cellular life and is required for many neurological processes, including the formation, updating and extinction of long-term memories. Protein synthesis is impaired in neurodegenerative diseases including tauopathies, in which pathology is caused by aberrant changes to the microtubule-associated protein tau. We recently showed that both global de novo protein synthesis and the synthesis of select ribosomal proteins (RPs) are decreased in mouse models of frontotemporal dementia (FTD) which express mutant forms of tau. However, a comprehensive analysis of the effect of FTD-mutant tau on ribosomes is lacking. Here we used polysome profiling, de novo protein labelling and mass spectrometry-based proteomics to examine how ribosomes are altered in models of FTD. We identified 10 RPs which were decreased in abundance in primary neurons taken from the K3 mouse model of FTD. We further demonstrate that expression of human tau (hTau) decreases both protein synthesis and biogenesis of the 60S ribosomal subunit, with these effects being exacerbated in the presence of FTD-associated tau mutations. Lastly, we demonstrate that expression of the amino-terminal projection domain of hTau is sufficient to reduce protein synthesis and ribosomal biogenesis. Together, these data reinforce a role for tau in impairing ribosomal function. Supplementary Information The online version contains supplementary material available at 10.1186/s40478-021-01208-4.

Eukaryotic ribosome biogenesis is an elaborate process during which ribosomal proteins assemble with the pre-rRNA while it is being processed and folded. Hundreds of assembly factors (AF) are required and transiently recruited to assist the sequential remodeling events. One of the most intricate ones is the stepwise removal of the internal transcribed spacer 2 (ITS2), between the 5.8S and 25S rRNAs, that constitutes together with five AFs the pre-60S ‘foot’. In the transition from nucleolus to nucleoplasm, Nop53 replaces Erb1 at the basis of the foot and recruits the RNA exosome for the ITS2 cleavage and foot disassembly. Here we comprehensively analyze the impact of Nop53 recruitment on the pre-60S compositional changes. We show that depletion of Nop53, different from nop53 mutants lacking the exosome-interacting motif, not only causes retention of the unprocessed foot in late pre-60S intermediates but also affects the transition from nucleolar state E particle to subsequent nuclear stages. Additionally, we reveal that Nop53 depletion causes the impairment of late maturation events such as Yvh1 recruitment. In light of recently described pre-60S cryo-EM structures, our results provide biochemical evidence for the structural role of Nop53 rearranging and stabilizing the foot interface to assist the Nog2 particle formation.

SummarySince multiple ribosomes can engage a single mRNA, nonuniform ribosome progression can result in collisions. Ribosome collisions during translation elongation elicit a multifaceted ribosome-associated quality control (RQC) response. Despite advanced mechanistic understanding of translation initiation, a parallel RQC pathway that acts on collided preinitiation complexes has not been described. Here, we show that blocking progression of scanning or elongating ribosomes past the start codon triggers uS3 and uS5 ribosomal ubiquitylation. We demonstrate that conditions that activate the integrated stress response can also induce preinitiation complex collisions. The ubiquitin ligase, RNF10, and the deubiquitylating enzyme, USP10, are the key regulators of uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in an autophagy-independent manner. This study identifies a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on pervasive ribosome collisions during translation initiation to modulate translation activity and capacity.

Human ribosomes, made of around 80 ribosomal proteins (RPs) and four ribosomal RNAs, have long been thought as uniform passive protein-making factories with little regulatory function. Recently, accumulating evidence showed heterogeneity of RP composition in ribosomes responsible for regulating gene expression in development and tumorigenesis. However, a comprehensive understanding of regulatory spectrum of RPs is still lacking. In this study, we conducted a systematic survey of regulatory specificity of human RPs on global gene expression. We quantified translational and transcriptional changes of gene expression upon deficiency of 75 RPs, including 44 from the large subunit (60S) and 31 from the small subunit (40S), by ribosomal profiling and RNA sequencing analysis. We showed that RP deficiency induced diverse expression changes, particularly at the translational level. RPs were subjected to co-translational regulation under ribosomal stress where deficiency of the 60S and the 40S RPs had opposite effects on the two subunits. RP deficiency perturbed expression of genes related to cell cycle, cellular metabolism, signal transduction and development. Deficiency of RPs from the 60S led to a greater repression effect on cell growth than that from the 40S by perturbing P53 signaling and cell cycle pathways. To demonstrate functional specificity of RPs, we showed that RPS8 deficiency stimulated cellular apoptosis and RPL13 or RPL18 deficiency promoted cellular senescence. We also showed that RPL11 and RPL15 played important roles in retina development and angiogenesis, respectively. Overall, our study demonstrated a widespread regulatory role of RPs in controlling cellular activity, providing an important resource which can offer novel insights into ribosome regulation in human diseases and cancer.

During their maturation, nascent 40S subunits enter a translation-like quality control cycle, where they are joined by mature 60S subunits to form 80S-like ribosomes. While these assembly intermediates are essential for maturation and quality control, how they form, and how their structure promotes quality control remains unknown. To address these questions, we determined the structure of an 80S-like ribosome assembly intermediate to an overall resolution of 3.4 Å. The structure, validated by biochemical data, resolves a large body of previously paradoxical data and illustrates how assembly and translation factors cooperate to promote the formation of an interface that lacks many mature subunit contacts but is stabilized by the universally conserved Dim1. We also show how Tsr1 enables this interface by blocking the canonical binding of eIF5B to 40S subunits, while maintaining its binding to 60S. The structure also shows how this interface leads to unfolding of the platform, which allows for temporal regulation of the ATPase Fap7, thus linking 40S maturation to quality-control during ribosome assembly.

Ribosome stalling at tandem CGA codons or poly(A) sequences activates quality controls for nascent polypeptides including ribosome-associated quality control (RQC) and no-go mRNA decay (NGD). In RQC pathway, Hel2-dependent uS10 ubiquitination and the RQC-trigger (RQT) complex are essential for subunit dissociation, and Ltn1-dependent ubiquitination of peptidyl-tRNA in the 60S subunit requires Rqc2. Here, we report that polytryptophan sequences induce Rqc2-independent RQC. More than 11 consecutive tryptophan residues induced RQC in a manner dependent on Hel2-mediated ribosome ubiquitination and the RQT complex. Polytryptophan sequence-mediated RQC was not coupled with CAT-tailing, and Rqc2 was not required for Ltn1-dependent degradation of the arrest products. Eight consecutive tryptophan residues located at the region proximal to the peptidyl transferase center in the ribosome tunnel inhibited CAT-tailing by tandem CGA codons. Polytryptophan sequences also induced Hel2-mediated canonical RQC-coupled NGD and RQC-uncoupled NGD outside the stalled ribosomes. We propose that poly-tryptophan sequences induce Rqc2-independent RQC, suggesting that CAT-tailing in the 60S subunit could be modulated by the polypeptide in the ribosome exit tunnel.

Eukaryotic ribosomes consist of a small 40S and a large 60S subunit that are assembled in a highly coordinated manner. More than 200 factors ensure correct modification, processing and folding of ribosomal RNA and the timely incorporation of ribosomal proteins1,2. Small subunit maturation ends in the cytosol, when the final rRNA precursor, 18S-E, is cleaved at site 3 by the endonuclease NOB13. Previous structures of human 40S precursors have shown that NOB1 is kept in an inactive state by its partner PNO14. The final maturation events, including the activation of NOB1 for the decisive rRNA-cleavage step and the mechanisms driving the dissociation of the last biogenesis factors have, however, remained unresolved. Here we report five cryo-electron microscopy structures of human 40S subunit precursors, which describe the compositional and conformational progression during the final steps of 40S assembly. Our structures explain the central role of RIOK1 in the displacement and dissociation of PNO1, which in turn allows conformational changes and activation of the endonuclease NOB1. In addition, we observe two factors, eukaryotic translation initiation factor 1A domain-containing protein (EIF1AD) and leucine-rich repeat-containing protein 47 (LRRC47), which bind to late pre-40S particles near RIOK1 and the central rRNA helix 44. Finally, functional data shows that EIF1AD is required for efficient assembly factor recycling and 18S-E processing. Our results thus enable a detailed understanding of the last steps in 40S formation in human cells and, in addition, provide evidence for principal differences in small ribosomal subunit formation between humans and the model organism Saccharomyces cerevisiae. Studies of five cryo-electron microscopy structures reveal the composition and conformational progression in the final maturation events of human 40S ribosomal subunit assembly.

Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 A resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 A showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.

Eukaryotic cells have evolved multiple mechanisms to detect and eliminate aberrant polypeptides. Co-translational protein surveillance systems play an important role in these mechanisms. These systems include ribosome-associated protein quality control (RQC) that detects aberrant nascent chains stalled on ribosomes and promotes their ubiquitination and degradation by the proteasome, and ribosome-associated chaperone Ssb/RAC, which ensures correct nascent chain folding. Despite the known function of RQC and Ssb/ribosome-associated complex (RAC) in monitoring the quality of newly generated polypeptides, whether they cooperate during initial stages of protein synthesis remains unexplored. Here, we provide evidence that Ssb/RAC and the ubiquitin ligase Ltn1, the major component of RQC, display genetic and functional cooperativity. Overexpression of Ltn1 rescues growth suppression of the yeast strain-bearing deletions of SSB genes during proteotoxic stress. Moreover, Ssb/RAC promotes Ltn1-dependent ubiquitination of nascent chains associated with 80S ribosomal particles but not with translating ribosomes. Consistent with this finding, quantitative western blot analysis revealed lower levels of Ltn1 associated with 80S ribosomes and with free 60S ribosomal subunits in the absence of Ssb/RAC. We propose a mechanism in which Ssb/RAC facilitates recruitment of Ltn1 to ribosomes, likely by detecting aberrations in nascent chains and leading to their ubiquitination and degradation.

Ribosome biogenesis is an elaborate and energetically expensive program that involve two hundred protein factors in eukaryotes. Nuclear export of pre-ribosomal particles is one central step which also serves as an internal structural checkpoint to ensure the proper completion of nuclear assembly events. Here we present four structures of human pre-60S particles isolated through a nuclear export factor NMD3, representing assembly stages immediately before and after nuclear export. These structures reveal locations of a dozen of human factors, including an uncharacterized factor TMA16 localized between the 5S RNA and the P0 stalk. Comparison of these structures shows a progressive maturation for the functional regions, such as peptidyl transferase centre and peptide exit tunnel, and illustrate a sequence of factor-assisted rRNA maturation events. These data facilitate our understanding of the global conservation of ribosome assembly in eukaryotes and species-specific features of human assembly factors., Ribosome biogenesis in eukaryotes is a complex process that involves more than 200 protein factors. Here the authors present a structural analysis of a collection of human pre-60S structures sampled through a nuclear export adaptor NMD3, representing structural snapshots of pre-60S particles immediately before and after passing through nuclear pore complex.

The recycling of ribosomes at stop codons for use in further rounds of translation is critical for efficient protein synthesis. Removal of the 60S subunit is catalyzed by the ATPase Rli1 (ABCE1) while removal of the 40S is thought to require Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR). However, it remains unclear how these Tma proteins cause 40S removal and control reinitiation of downstream translation. Here we used a 40S ribosome footprinting strategy to directly observe intermediate steps of ribosome recycling in cells. Deletion of the genes encoding these Tma proteins resulted in broad accumulation of unrecycled 40S subunits at stop codons, directly establishing their role in 40S recycling. Furthermore, the Tma20/Tma22 heterodimer was responsible for a majority of 40S recycling events while Tma64 played a minor role. Introduction of an autism-associated mutation into TMA22 resulted in a loss of 40S recycling activity, linking ribosome recycling and neurological disease., Following the termination of translation at stop codons, the eukaryotic 60S subunit of the ribosome is removed by the ATPase ABCE1. Here using 40S ribosome footprinting the authors provide a direct demonstration that the yeast orthologs of eIF2D, MCT-1, and DENR recycle the 40S subunits.

A long-standing problem is how cells that lack one of the highly similar ribosomal proteins (RPs) often display distinct phenotypes. Yeast and other organisms live longer when they lack specific ribosomal proteins, especially of the large 60S subunit of the ribosome. However, longevity is neither associated with the generation time of RP deletion mutants nor with bulk inhibition of protein synthesis. Here, we queried actively dividing RP mutants through the cell cycle. Our data link transcriptional, translational, and metabolic changes to phenotypes associated with the loss of paralogous RPs. We uncovered translational control of transcripts encoding enzymes of methionine and serine metabolism, which are part of one-carbon (1C) pathways. Cells lacking Rpl22Ap, which are long-lived, have lower levels of metabolites associated with 1C metabolism. Loss of 1C enzymes increased the longevity of wild type cells. 1C pathways exist in all organisms and targeting the relevant enzymes could represent longevity interventions.

A long-standing problem is how cells that lack one of the highly similar ribosomal proteins (RPs) often display distinct phenotypes. Some may reflect general effects due to lower growth rate and ribosome levels, but a number of diverse phenotypes cannot be explained through this mechanism. Yeast and other organisms live longer when they lack specific ribosomal proteins, especially of the large 60S subunit of the ribosome. However, longevity is neither associated with the generation time of RP deletion mutants nor with bulk inhibition of protein synthesis. Here, we comprehensively queried actively dividing RP paralog mutants through the cell cycle. Our data link transcriptional, translational, and metabolic changes to phenotypes associated with the loss of paralogous RPs. We uncovered specific translational control of transcripts encoding enzymes of methionine and serine metabolism, which are part of one-carbon (1C) pathways. Cells lacking Rpl22Ap, which are long-lived, have lower levels of metabolites associated with 1C metabolism. Loss of 1C enzymes, such as the serine hydroxymethyltransferase Shm2p increased the longevity of wild type cells. These results provide a molecular basis for paralog-specific phenotypes in ribosomal mutants and underscore the significance of 1C metabolic pathways in mechanisms of cell division and cellular aging. 1C pathways exist in all organisms, including humans, and targeting the relevant enzymes could represent longevity interventions.

An important but often overlooked aspect of gene regulation occurs at the level of protein translation. Many genes are regulated not only by transcription but by their propensity to be recruited to actively translating ribosomes (polysomes). Polysome profiling allows for the separation of unbound 40S and 60S subunits, 80S monosomes, and actively translating mRNA bound by two or more ribosomes. Thus, this technique allows for actively translated mRNA to be isolated. Transcript abundance can then be compared between actively translated mRNA and all mRNA present in a sample to identify instances of post-transcriptional regulation. Additionally, polysome profiling can be used as a readout of global translation rates by quantifying the proportion of actively translating ribosomes within a sample. Previously established protocols for polysome profiling rely on the absorbance of RNA to visualize the presence of polysomes within the fractions. However, with the advent of flow cells capable of detecting fluorescence, the association of fluorescently tagged proteins with polysomes can be detected and quantified in addition to the absorbance of RNA. This protocol provides detailed instructions on how to perform fluorescent polysome profiling in C. elegans to collect actively translated mRNA, to quantify changes in global translation, and to detect ribosomal binding partners.

Inhibition of the synthesis of a ribosomal protein (r-protein) abrogates the assembly of its cognate subunit, while assembly of the other subunit continues. Ribosomal components that are not stably incorporated into ribosomal particles due to the disrupted assembly are rapidly degraded. The 60S protein uL18/L5 is an exception, because this protein accumulates extra-ribosomally during inhibition of 60S assembly. Since the r-proteins in each ribosomal subunit are essential only for formation of their own subunit, it would be predicted that accumulation of extra-ribosomal uL18/5 only occurs during restriction of 60S assembly, and not during abolition of 40S assembly. Contrary to this prediction, we report here that repression of 40S r-protein genes does in fact lead to accumulation of uL18/L5 does outside the ribosome due modified 60S assembly. Furthermore, the effect varies depending on which 40S ribosomal protein is repressed. We propose that disruption of early steps in the 40S subunit assembly changes the kinetics of 60S subunit assembly resulting in a buildup of extra-ribosomal uL18/L5, even though 60S formation continues. Finally, our results show that maintenance of the pool of extra-ribosomal uL18/L5 requires continual protein synthesis showing that extra-ribosomal protein is not stable, but is slowly “consumed” by incorporation into 60S subunits and/or turnover.

The ribosomal 40S subunit is composed of 18S ribosomal RNA (rRNA) and 33 ribosomal proteins (r‐proteins). This number also includes ribosome‐associated receptor for activating C kinase 1 (RACK1) (Pisarev et al., 2008; Sengupta et al., 2004). The 60S subunit has 5S, 5.8S and 25‐26S rRNAs, and 48 r‐proteins. The translation of r‐proteins occurs in the cytoplasm and, subsequently, they are transported into the nucleolus for subunit assembly. Final maturation takes place after export to the cytoplasm, where the acidic P‐proteins P0, P1, P2 and P3 bind to the 60S subunit to form a motile stalk (Szick et al., 1998). The two subunits combine during translation initiation to form a translation‐competent 80S ribosome. Polysomes consist of varying numbers of 80S ribosomes, which all synthesize the same protein. Global down‐regulation of translation initiation, which causes alterations in the abundance of 40S and 60S subunits, monosomes and polysomes, may occur in various stress conditions (Bailey‐Serres et al., 2009). In plants, multiple paralogous r‐protein genes that share high sequence similarity encode each r‐protein (Barakat et al., 2001; Hummel et al., 2015; Whittle and Krochko, 2009). The majority of them are transcribed (Barakat et al., 2001; Whittle and Krochko, 2009), translated (Schippers and Mueller‐Roeber, 2010) and incorporated into ribosomes (Carroll et al., 2008; Chang et al., 2005; Giavalisco et al., 2005; Hummel et al., 2012, 2015). The A. thaliana genome contains 242 functional r‐protein genes (Barakat et al., 2001; Chang et al., 2005; Hummel et al., 2015). Their incorporation into ribosomes would allow ~1034 compositionally different ribosome assemblies (Hummel et al., 2012). Transcribed r‐protein genes vary with the tissue type and developmental stage (McIntosh and Bonham‐Smith, 2006; Whittle and Krochko, 2009), as well as with external stimuli (Wang et al., 2013). Importantly, mutation and knockdown studies indicate that certain paralogues may have specialized functions in plant growth and development (Degenhardt and Bonham‐Smith, 2008; Horiguchi et al., 2012; Schippers and Mueller‐Roeber, 2010). The r‐proteins of A. thaliana range from 3.4 to 44.7 kDa in size (Barakat et al., 2001; Chang et al., 2005). The r‐proteins are mostly located on the surface of ribosomes (Klinge et al., 2012). It has been suggested that they serve as a docking station for ribosome‐associated proteins that assist in translation regulation (Xue and Barna, 2012). Accordingly, minor changes in the ribosome surfaces caused by heterogeneity in the r‐protein sequences could affect the repertoire of ribosome‐associated proteins bound on the ribosome surfaces. Proteomic studies of ribosomes have identified non‐ribosomal proteins that co‐purify with cytosolic plant ribosomes (Carroll et al., 2008; Chang et al., 2005; Hummel et al., 2012). For example, the well‐known RACK1 binds to the 40S subunit in a 1 : 1 ratio and interacts with proteins involved in translational regulation (Adams et al., 2011; Kundu et al., 2013; Nilsson et al., 2004). In this work, we studied the protein composition of ribosomes of N. benthamiana. N. benthamiana is widely used as a model plant to study plant–microbe interactions (Goodin et al., 2008), as it is readily infected with numerous plant pathogens, including viruses, bacteria and fungi. We also investigated the effects of two plant pathogens, A. tumefaciens and Potato virus A (PVA, family Potyviridae), on N. benthamiana riboproteome and translational activity. Potyviruses form a large group of positive‐stranded RNA viruses (reviewed in Ivanov et al., 2014). Potyvirus infections induce the transcription of r‐protein genes (Alfenas‐Zerbini et al., 2009; Dardick, 2007; Yang et al., 2007), suggesting that specialized ribosomes or certain r‐proteins might be required during infection. A. tumefaciens is a Gram‐negative soil bacterium belonging to the family Rhizobiaceae (reviewed in Tarkowski and Vereecke, 2014). In contrast with potyviruses, A. tumefaciens infection represses r‐protein expression (Ditt et al., 2006). The proteomic investigation and annotation of healthy, A. tumefaciens‐ and PVA‐infected N. benthamiana ribosomes, which are reported here, will be an important source of information for further studies of translational control in pathogen‐infected plants.

Ribosome biosynthesis, best studied in opisthokonts, is a highly complex process involving numerous protein and RNA factors. Yet, very little is known about the early stages of pre-18S rRNA processing even in these model organisms, let alone the conservation of this mechanism in other eukaryotes. Here we extend our knowledge of this process by identifying and characterizing the essential protein TbUTP10, a homolog of yeast U3 small nucleolar RNA-associated protein 10 - UTP10 (HEATR1 in human), in the excavate parasitic protist Trypanosoma brucei. We show that TbUTP10 localizes to the nucleolus and that its ablation by RNAi knock-down in two different T. brucei life cycle stages results in similar phenotypes: a disruption of pre-18S rRNA processing, exemplified by the accumulation of rRNA precursors, a reduction of mature 18S rRNA, and also a decrease in the level of U3 snoRNA. Moreover, polysome profiles of the RNAi-induced knock-down cells show a complete disappearance of the 40S ribosomal subunit, and a prominent accumulation of the 60S large ribosomal subunit, reflecting impaired ribosome assembly. Thus, TbUTP10 is an important protein in the processing of 18S rRNA.

Ribosome biogenesis involves numerous pre-rRNA processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Biedka et al. show that ribosomal proteins and assembly factors remodel several neighborhoods, including two 60S ribosomal subunit functional centers, during removal of the ITS2 spacer RNA., Ribosome biogenesis involves numerous preribosomal RNA (pre-rRNA) processing events to remove internal and external transcribed spacer sequences, ultimately yielding three mature rRNAs. Removal of the internal transcribed spacer 2 spacer RNA is the final step in large subunit pre-rRNA processing and begins with endonucleolytic cleavage at the C2 site of 27SB pre-rRNA. C2 cleavage requires the hierarchical recruitment of 11 ribosomal proteins and 14 ribosome assembly factors. However, the function of these proteins in C2 cleavage remained unclear. In this study, we have performed a detailed analysis of the effects of depleting proteins required for C2 cleavage and interpreted these results using cryo–electron microscopy structures of assembling 60S subunits. This work revealed that these proteins are required for remodeling of several neighborhoods, including two major functional centers of the 60S subunit, suggesting that these remodeling events form a checkpoint leading to C2 cleavage. Interestingly, when C2 cleavage is directly blocked by depleting or inactivating the C2 endonuclease, assembly progresses through all other subsequent steps.

Assembly of eukaryotic ribosome is a complicated and dynamic process that involves a series of intermediates. It is unknown how the highly intertwined structure of 60S large ribosomal subunits is established. Here, we report the structure of an early nucleolar pre-60S ribosome determined by cryo-electron microscopy at 3.7 Å resolution, revealing a half-assembled subunit. Domains I, II and VI of 25S/5.8S rRNA pack tightly into a native-like substructure, but domains III, IV and V are not assembled. The structure contains 12 assembly factors and 19 ribosomal proteins, many of which are required for early processing of large subunit rRNA. The Brx1-Ebp2 complex would interfere with the assembly of domains IV and V. Rpf1, Mak16, Nsa1 and Rrp1 form a cluster that consolidates the joining of domains I and II. Our structure reveals a key intermediate on the path to establishing the global architecture of 60S subunits. Electronic supplementary material The online version of this article (10.1007/s13238-018-0526-7) contains supplementary material, which is available to authorized users.

YcfD from Escherichia coli is a homologue of the human ribosomal oxygenases NO66 and MINA53, which catalyse histidyl-hydroxylation of the 60S subunit and affect cellular proliferation (Ge et al., Nat Chem Biol 12:960–962, 2012). Bioinformatic analysis identified a potential homologue of ycfD in the thermophilic bacterium Rhodothermus marinus (ycfDRM). We describe studies on the characterization of ycfDRM, which is a functional 2OG oxygenase catalysing (2S,3R)-hydroxylation of the ribosomal protein uL16 at R82, and which is active at significantly higher temperatures than previously reported for any other 2OG oxygenase. Recombinant ycfDRM manifests high thermostability (Tm 84 °C) and activity at higher temperatures (Topt 55 °C) than ycfDEC (Tm 50.6 °C, Topt 40 °C). Mass spectrometric studies on purified R. marinus ribosomal proteins demonstrate a temperature-dependent variation in uL16 hydroxylation. Kinetic studies of oxygen dependence suggest that dioxygen availability can be a limiting factor for ycfDRM catalysis at high temperatures, consistent with incomplete uL16 hydroxylation observed in R. marinus cells. Overall, the results that extend the known range of ribosomal hydroxylation, reveal the potential for ycfD-catalysed hydroxylation to be regulated by temperature/dioxygen availability, and that thermophilic 2OG oxygenases are of interest from a biocatalytic perspective. Electronic supplementary material The online version of this article (10.1007/s00792-018-1016-9) contains supplementary material, which is available to authorized users.

Cryo-electron microscopy structures of multiple states along the assembly pathway of the nucleolar pre-60S ribosomal subunit suggest that folding of the pre-ribosomal RNA and sequential binding of transient assembly factors are regulated by steric hindrance among these factors. Cryo-electron microscopy approaches provide unprecedented views of how large molecular complexes assemble in cells. Sebastian Klinge and colleagues used these techniques to analyse the formation of the large pre-60S ribosomal subunit at a resolution of 3.4 A. These analyses show that the concerted activity of several assembly factors is important for the stabilization and remodelling of intermediate structures of RNA and proteins while avoiding premature folding states. Progress through sequential steps of this assembly pathway is needed to achieve the correct conformation of the polypeptide exit tunnel of the ribosome. Early co-transcriptional events during eukaryotic ribosome assembly result in the formation of precursors of the small (40S) and large (60S) ribosomal subunits1. A multitude of transient assembly factors regulate and chaperone the systematic folding of pre-ribosomal RNA subdomains. However, owing to a lack of structural information, the role of these factors during early nucleolar 60S assembly is not fully understood. Here we report cryo-electron microscopy (cryo-EM) reconstructions of the nucleolar pre-60S ribosomal subunit in different conformational states at resolutions of up to 3.4 A. These reconstructions reveal how steric hindrance and molecular mimicry are used to prevent both premature folding states and binding of later factors. This is accomplished by the concerted activity of 21 ribosome assembly factors that stabilize and remodel pre-ribosomal RNA and ribosomal proteins. Among these factors, three Brix-domain proteins and their binding partners form a ring-like structure at ribosomal RNA (rRNA) domain boundaries to support the architecture of the maturing particle. The existence of mutually exclusive conformations of these pre-60S particles suggests that the formation of the polypeptide exit tunnel is achieved through different folding pathways during subsequent stages of ribosome assembly. These structures rationalize previous genetic and biochemical data and highlight the mechanisms that drive eukaryotic ribosome assembly in a unidirectional manner.

The contribution of most ribosomal proteins to ribo- some synthesis has been quite well analysed in Sac- charomyces cerevisiae . However, few yeast riboso- mal proteins still await characterization. Herein, we show that L14, an essential 60S ribosomal protein, assembles in the nucleolus at an early stage into pre- 60S particles. Depletion of L14 results in a deficit in 60S subunits and defective processing of 27SA 2 and 27SA 3 to 27SB pre-rRNAs. As a result, 27S pre-rRNAs are subjected to turnover and export of pre-60S par- ticles is blocked. These phenotypes likely appear as the direct consequence of the reduced pre-60S par- ticle association not only of L14 upon its depletion but also of a set of neighboring ribosomal proteins located at the solvent interface of 60S subunits and the adjacent region surrounding the polypeptide exit tunnel. These pre-60S intermediates also lack some essential trans -acting factors required for 27SB pre- rRNA processing but accumulate practically all fac- tors required for processing of 27SA 3 pre-rRNA. We have also analysed the functional interaction be- tween the eukaryote-specific carboxy-terminal exten- sions of the neighboring L14 and L16 proteins. Our results indicate that removal of the most distal parts of these extensions cause slight translation alter- ations in mature 60S subunits. Ministerio de Economía y Competitividad (BFU2013-42958-P), (BFU2016-75352- P) Deutsche Forschungs- gemeinschaft’ (SFB 960)

In the ribosome-associated quality control (RQC) pathway, stalled ribosomes are ubiquitinated and dissociated into subunits. The nascent protein chain associated with the 60S ribosomal subunit is ubiquitinated by the E3 ligase Listerin (Ltn1) and is released from tRNA by ANKZF1 (Vms1) for proteasomal degradation. Shao and colleagues now report that ANKZF1 (Vms1)-cleaved tRNAs are recycled via a two-step process that requires the removal of a terminal 2′,3′-cyclic phosphate and the addition of CCA by TRNT1.

Summary Impaired ribosome function is the underlying etiology in a group of bone marrow failure syndromes called ribosomopathies. However, how ribosomes are regulated remains poorly understood, as are approaches to restore hematopoietic stem cell (HSC) function loss because of defective ribosome biogenesis. Here we reveal a role of the E3 ubiquitin ligase HectD1 in regulating HSC function via ribosome assembly and protein translation. Hectd1-deficient HSCs exhibit a striking defect in transplantation ability and ex vivo maintenance concomitant with reduced protein synthesis and growth rate under stress conditions. Mechanistically, HectD1 ubiquitinates and degrades ZNF622, an assembly factor for the ribosomal 60S subunit. Hectd1 loss leads to accumulation of ZNF622 and the anti-association factor eIF6 on 60S, resulting in 60S/40S joining defects. Importantly, Znf622 depletion in Hectd1-deficient HSCs restored ribosomal subunit joining, protein synthesis, and HSC reconstitution capacity. These findings highlight the importance of ubiquitin-coordinated ribosome assembly in HSC regeneration.

The ability to detect and respond to oxidative stress is crucial to the survival of living organisms. In cells, sensing of increased levels of reactive oxygen species (ROS) activates many defensive mechanisms that limit or repair damage to cell components. The ROS-signaling responses necessary for cell survival under oxidative stress conditions remain incompletely understood, especially for the translational machinery. Here, we found that drug treatments or a genetic deficiency in the thioredoxin system that increase levels of endogenous hydrogen peroxide in the yeast Saccharomyces cerevisiae promote site-specific endonucleolytic cleavage in 25S ribosomal RNA (rRNA) adjacent to the c loop of the expansion segment 7 (ES7), a putative regulatory region located on the surface of the 60S ribosomal subunit. Our data also show that ES7c is cleaved at early stages of the gene expression program that enables cells to successfully counteract oxidative stress and is not a prerequisite or consequence of apoptosis. Moreover, the 60S subunits containing ES7c-cleaved rRNA cofractionate with intact subunits in sucrose gradients and repopulate polysomes after a short starvation-induced translational block, indicating their active role in translation. These results demonstrate that ES7c cleavage in rRNA is an early and sensitive marker of increased ROS levels in yeast cells and suggest that changes in ribosomes may be involved in the adaptive response to oxidative stress.

Ribosomes routinely prepared from Escherichia coli strain K12 contain intact (70 amino acids) and short (62 amino acids) forms of ribosomal protein L31. By contrast, ribosomes prepared from ompT mutant cells, which lack protease 7, contain only intact L31, suggesting that L31 is cleaved by protease 7 during ribosome preparation. We compared ribosomal subunit association in wild-type and ompT − strains. In sucrose density gradient centrifugation under low Mg2+, 70S content was very high in ompT − ribosomes, but decreased in the wild-type ribosomes containing short L31. In addition, ribosomes lacking L31 failed to associate ribosomal subunits in low Mg2+. Therefore, intact L31 is required for subunit association, and the eight C-terminal amino acids contribute to the association function. In vitro translation was assayed using three different systems. Translational activities of ribosomes lacking L31 were 40% lower than those of ompT − ribosomes with one copy of intact L31, indicating that L31 is involved in translation. Moreover, in the stationary phase, L31 was necessary for 100S formation. The strain lacking L31 grew very slowly. A structural analysis predicted that the L31 protein spans the 30S and 50S subunits, consistent with the functions of L31 in 70S association, 100S formation, and translation.

Ribosomes are responsible for translating the genome, in the form of mRNA, into the proteome in all organisms. Biogenesis of ribosomes in eukaryotes is a complex process involving numerous remodeling events driven in part by the concerted actions of hundreds of protein assembly factors. A major challenge in studying eukaryotic ribosome assembly has, until recently, been a lack of structural data to facilitate understanding of the conformational and compositional changes the pre-ribosome undergoes during its construction. Cryo-electron microscopy (cryo-EM) has begun filling these gaps; recent advances in cryo-EM have enabled the determination of several high resolution pre-ribosome structures. This review focuses mainly on lessons learned from the study of pre-60S particles purified from yeast using the assembly factor Nog2 as bait. These Nog2 particles provide insight into many aspects of nuclear stages of 60S subunit assembly, including construction of major 60S subunit functional centers and processing of the ITS2 spacer RNA.

The biogenesis of ribosomes is a central process in all dividing cells. Eukaryotic ribosomes are composed of a large 60S and a small 40S subunit, each comprising a complex assembly of ribosomal RNA (rRNA) and ribosomal proteins (r-proteins). The synthesis of these constituents is spatially separated, with r-proteins being produced by translation in the cytoplasm, while rRNA is generated by transcription in the nucleus. Hence, the arrangement of r-proteins and rRNA into large ribonucleoprotein complexes requires dedicated mechanisms ensuring their encounter in the same compartment. To this end, r-proteins need to be safely delivered to the nucleus where they assemble with the rRNA. Beyond these initial challenges, the synthesis of ribosomes does not merely comprise the joining of r-proteins with rRNA, but occurs in a complex assembly line involving multiple maturation steps, including the processing and folding of rRNA. R-proteins usually have composite rRNA binding sites, with several different rRNA helices contributing to the full interaction. Not all of these interaction sites may already be accessible at the point when an r-protein is incorporated, necessitating that some of the r-protein-rRNA contacts are formed at later maturation stages. In our two recent studies, we investigated the ribosome assembly path of r-proteins in the yeast Saccharomyces cerevisiae using the small subunit r-protein S3 (Rps3) as a model. Our studies revealed intricate mechanisms to protect the protein, transport it into the nucleus, integrate it into pre-ribosomal precursor particles and promote its final stable association with 40S subunits.

© 2019 Ramos-Sáenz et al., In Saccharomyces cerevisiae, more than 250 trans-acting factors are involved in the maturation of 40S and 60S ribosomal subunits. The expression of most of these factors is transcriptionally coregulated to ensure correct ribosome production under a wide variety of environmental and intracellular conditions. Here, we identified the essential nucleolar Pol5 protein as a novel trans-acting factor required for the synthesis of 60S ribosomal subunits. Pol5 weakly and/or transiently associates with early to medium pre-60S ribosomal particles. Depletion of and temperature-sensitive mutations in Pol5 result in a deficiency of 60S ribosomal subunits and accumulation of half-mer polysomes. Both processing of 27SB pre-rRNA to mature 25S rRNA and release of pre-60S ribosomal particles from the nucle(ol)us to the cytoplasm are impaired in the Pol5-depleted strain. Moreover, we identified the genes encoding ribosomal proteins uL23 and eL27A as multicopy suppressors of the slow growth of a temperature-sensitive pol5 mutant. These results suggest that Pol5 could function in ensuring the correct folding of 25S rRNA domain III; thus, favoring the correct assembly of these two ribosomal proteins at their respective binding sites into medium pre-60S ribosomal particles. Pol5 is homologous to the human tumor suppressor Myb-binding protein 1A (MYBBP1A). However, expression of MYBBP1A failed to complement the lethal phenotype of a pol5 null mutant strain though interfered with 60S ribosomal subunit biogenesis., This work was supported by the Spanish Ministry of Economy and Competitiveness (MINECO) and ERDF to J.d.l.C. (BFU2016-75352-P AEI/FEDER, EU) and to M.D. (BFU2014-52729-P, BFU2017-88192-P). D.G.-A. is a recipient of a contract of the Youth Employment Initiative from the University of Seville/Junta de Andalucía/European Social Fund.

Ribosomal proteins are highly expressed, and the quality of ribosomal proteins must be rigorously controlled to build up a functional ribosome. Rpl43, ribosomal protein large subunit 43, is located nearby the E-site of ribosomes. In our previous study, we found that Puf6, Loc1, and Rpl43 form a trimeric complex in Saccharomyces cerevisiae. Rpl43 protein levels are under-accumulated in the absence of PUF6 or LOC1. However, why the loss of Puf6 or Loc1 decreased the protein levels of Rpl43 remained unclear. In the present study, we further dissected the connections among these three proteins and found that the processing defects of pre-ribosomal RNA in puf6&Delta, and loc1&Delta, are similar to those of the mutant with depletion of Rpl43. The stability of newly synthesized Rpl43 protein decreased slightly in puf6&Delta, and significantly in loc1&Delta, We also found that Puf6 and Loc1 could interact with nascent Rpl43 co-translationally via the N-terminus of Rpl43. While the association and dissociation of Rpl43 with karyopherins did not depend on Puf6 and Loc1, Puf6 and Loc1 interacted with nascent Rpl43 in collaboration. While the N-terminus of Puf6 contained nuclear localization signals for transport, the PUF (Pumilio) domain was essential to interaction with Loc1, Rpl43, and 60S subunits. The C-terminus of Loc1 is more important for interaction with Puf6 and Rpl43. In this study, we found that Puf6 and Loc1 are the dedicated chaperones of ribosomal protein Rpl43 and also analyzed the potential interaction domains among the three proteins. Correct formation of the Puf6, Loc1, and Rpl43 ternary complex is required to properly proceed to the next step in 60S biogenesis.