A translation-like cycle is a quality control checkpoint for maturing 40S ribosome subunits.
ABSTRACT: Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.
Project description:The recycling of ribosomal subunits after translation termination is critical for efficient gene expression. Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) function as 40S recycling factors in vitro, but it is unknown whether they perform this function in vivo. Ribosome profiling of tma deletion strains revealed 80S ribosomes queued behind the stop codon, consistent with a block in 40S recycling. We found that unrecycled ribosomes could reinitiate translation at AUG codons in the 3' UTR, as evidenced by peaks in the footprint data and 3' UTR reporter analysis. In vitro translation experiments using reporter mRNAs containing upstream open reading frames (uORFs) further established that reinitiation increased in the absence of these proteins. In some cases, 40S ribosomes appeared to rejoin with 60S subunits and undergo an 80S reinitiation process in 3' UTRs. These results support a crucial role for Tma64, Tma20, and Tma22 in recycling 40S ribosomal subunits at stop codons and translation reinitiation.
Project description:Late in their maturation, nascent small (40S) ribosomal subunits bind 60S subunits to produce 80S-like ribosomes. Because of the analogy of this translation-like cycle to actual translation, and because 80S-like ribosomes do not produce any protein, it has been suggested that this represents a quality control mechanism for subunit functionality. Here we use genetic and biochemical experiments to show that the essential ATPase Fap7 promotes formation of the rotated state, a key intermediate in translocation, thereby releasing the essential assembly factor Dim1 from pre-40S subunits. Bypassing this quality control step produces defects in reading frame maintenance. These results show how progress in the maturation cascade is linked to a test for a key functionality of 40S ribosomes: their ability to translocate the mRNA?tRNA pair. Furthermore, our data demonstrate for the first time that the translation-like cycle is a quality control mechanism that ensures the fidelity of the cellular ribosome pool.
Project description:Recent reports have increased our knowledge of the consecutive steps during 60S ribosome biogenesis substantially, but 40S subunit formation is less well understood. Here, we investigate the maturation of nucleolar 90S pre-ribosomes into cytoplasmic 40S pre-ribosomes. During the transition from 90S to 40S particles, the majority of non-ribosomal proteins (approximately 30 species) dissociate, and significantly fewer factors associate with 40S pre-ribosomes. Notably, some of these components are part of both early 90S and intermediate 40S pre-particles in the nucleolus (e.g. Enp1p, Dim1p and Rrp12p), whereas others (e.g. Rio2p and Nob1p) are found mainly on late cytoplasmic pre-40S subunits. Finally, temperature-sensitive mutants mapping either in earlier (enp1-1) or later (rio2-1) components exhibit defects in the formation and nuclear export of pre-40S subunits. Our data provide an initial biochemical map of the pre-40S ribosomal subunit on its path from the nucleolus to the cytoplasm. This pathway involves fewer changes in composition than seen during 60S biogenesis.
Project description:Hcr1/eIF3j is a sub-stoichiometric subunit of eukaryotic initiation factor 3 (eIF3) that can dissociate the post-termination 40S ribosomal subunit from mRNA in vitro. We examine this ribosome recycling role in vivo by ribosome profiling and reporter assays and find that loss of Hcr1 leads to reinitiation of translation in 3' UTRs, consistent with a defect in recycling. However, the defect appears to be in the recycling of the 60S subunit, rather than the 40S subunit, because reinitiation does not require an AUG codon and is suppressed by overexpression of the 60S dissociation factor Rli1/ABCE1. Consistent with a 60S recycling role, overexpression of Hcr1 cannot compensate for loss of 40S recycling factors Tma64/eIF2D and Tma20/MCT-1. Intriguingly, loss of Hcr1 triggers greater expression of RLI1 via an apparent feedback loop. These findings suggest Hcr1/eIF3j is recruited to ribosomes at stop codons and may coordinate the transition to a new round of translation.
Project description:The cricket paralysis virus intergenic region internal ribosomal entry site (CrPV IGR IRES) can assemble translation initiation complexes by binding to 40S subunits without Met-tRNA(Met)(i) and initiation factors (eIFs) and then by joining directly with 60S subunits, yielding elongation-competent 80S ribosomes. Here, we report that eIF1, eIF1A and eIF3 do not significantly influence IRES/40S subunit binding but strongly inhibit subunit joining and the first elongation cycle. The IRES can avoid their inhibitory effect by its ability to bind directly to 80S ribosomes. The IRES's ability to bind to 40S subunits simultaneously with eIF1 allowed us to use directed hydroxyl radical cleavage to map its position relative to the known position of eIF1. A connecting loop in the IRES's pseudoknot (PK) III domain, part of PK II and the entire domain containing PK I are solvent-exposed and occupy the E site and regions of the P site that are usually occupied by Met-tRNA(Met)(i).
Project description:During ribosome recycling, posttermination complexes are dissociated by ABCE1 and eRF1 into 60S and tRNA/mRNA-associated 40S subunits, after which tRNA and mRNA are released by eIF1/eIF1A, Ligatin, or MCT-1/DENR. Occasionally, 40S subunits remain associated with mRNA and reinitiate at nearby AUGs. We recapitulated reinitiation using a reconstituted mammalian translation system. The presence of eIF2, eIF3, eIF1, eIF1A, and Met-tRNAi(Met) was sufficient for recycled 40S subunits to remain on mRNA, scan bidirectionally, and reinitiate at upstream and downstream AUGs if mRNA regions flanking the stop codon were unstructured. Imposition of 3' directionality additionally required eIF4F. Strikingly, posttermination ribosomes were not stably anchored on mRNA and migrated bidirectionally to codons cognate to the P site tRNA. Migration depended on the mode of peptide release (puromycin > eRF1?eRF3) and nature of tRNA and was enhanced by eEF2. The mobility of posttermination ribosomes suggests that some reinitiation events could involve 80S ribosomes rather than 40S subunits.
Project description:Eukaryotic translation initiation factor eIF5B is a ribosome-dependent GTPase that mediates displacement of initiation factors from the 40S ribosomal subunit in 48S initiation complexes and joining of 40S and 60S subunits. Here, we determined eIF5B's position on 80S ribosomes by directed hydroxyl radical cleavage. In the resulting model, eIF5B is located in the intersubunit cleft of the 80S ribosome: domain 1 is positioned near the GTPase activating center of the 60S subunit, domain 2 interacts with the 40S subunit (helices 3, 5 and the base of helix 15 of 18S rRNA and ribosomal protein (rp) rpS23), domain 3 is sandwiched between subunits and directly contacts several ribosomal elements including Helix 95 of 28S rRNA and helix 44 of 18S rRNA, domain 4 is near the peptidyl-transferase center and its helical subdomain contacts rpL10E. The cleavage data also indicate that binding of eIF5B might induce conformational changes in both subunits, with ribosomal segments wrapping around the factor. Some of these changes could also occur upon binding of other translational GTPases, and may contribute to factor recognition.
Project description:During eukaryotic ribosome biogenesis, members of the conserved atypical serine/threonine protein kinase family, the RIO kinases (Rio1, Rio2 and Rio3) function in small ribosomal subunit biogenesis. Structural analysis of Rio2 indicated a role as a conformation-sensing ATPase rather than a kinase to regulate its dynamic association with the pre-40S subunit. However, it remained elusive at which step and by which mechanism the other RIO kinase members act. Here, we have determined the crystal structure of the human Rio1-ATP-Mg(2+) complex carrying a phosphoaspartate in the active site indicative of ATPase activity. Structure-based mutations in yeast showed that Rio1's catalytic activity regulates its pre-40S association. Furthermore, we provide evidence that Rio1 associates with a very late pre-40S via its conserved C-terminal domain. Moreover, a rio1 dominant-negative mutant defective in ATP hydrolysis induced trapping of late biogenesis factors in pre-ribosomal particles, which turned out not to be pre-40S but 80S-like ribosomes. Thus, the RIO kinase fold generates a versatile ATPase enzyme, which in the case of Rio1 is activated following the Rio2 step to regulate one of the final 40S maturation events, at which time the 60S subunit is recruited for final quality control check.
Project description:Following translation termination, ribosomal subunits dissociate to become available for subsequent rounds of protein synthesis. In many translation-inhibiting stress conditions, e.g. glucose starvation in yeast, free ribosomal subunits reassociate to form a large pool of non-translating 80S ribosomes stabilized by the 'clamping' Stm1 factor. The subunits of these inactive ribosomes need to be mobilized for translation restart upon stress relief. The Dom34-Hbs1 complex, together with the Rli1 NTPase (also known as ABCE1), have been shown to split ribosomes stuck on mRNAs in the context of RNA quality control mechanisms. Here, using in vitro and in vivo methods, we report a new role for the Dom34-Hbs1 complex and Rli1 in dissociating inactive ribosomes, thereby facilitating translation restart in yeast recovering from glucose starvation stress. Interestingly, we found that this new role is not restricted to stress conditions, indicating that in growing yeast there is a dynamic pool of inactive ribosomes that needs to be split by Dom34-Hbs1 and Rli1 to participate in protein synthesis. We propose that this provides a new level of translation regulation.
Project description:The recycling of ribosomal subunits after translation termination is critical for efficient gene expression. Tma64 (eIF2D), Tma20 (MCT-1), and Tma22 (DENR) function as 40S recycling factors in vitro, but it is unknown whether they perform this function in vivo or serve as alternative initiation factors. Ribosome profiling of strains missing these factors revealed 80S ribosomes queued behind the stop codon, consistent with a block in 40S recycling. We found that unrecycled ribosomes could reinitiate translation at AUG codons in the 3’UTR, as evidenced by peaks in the footprint data and 3’UTR reporter analysis. In vitro translation experiments using reporter mRNAs containing upstream ORFs (uORFs) further established that reinitiation increased in the absence of these proteins. In some cases, 40S ribosomes appeared to rejoin with 60S subunits and undergo an alternative 80S reinitiation process in 3’UTRs. These results support a crucial role for Tma64, Tma20, and Tma22 in the recycling of 40S ribosomal subunits at stop codons and translation reinitiation. Overall design: 14 biological samples are included in the study for ribosome footprinting. These include wild-type and combination knockouts of TMA64, TMA20, and TMA22, and mutation of SUI1.