The ribosome-associated quality control (RQC) pathway degrades nascent chains (NCs) arising from interrupted translation. First, recycling factors split stalled ribosomes, yielding NC-tRNA/60S ...ribosome-nascent chain complexes (60S RNCs). 60S RNCs associate with NEMF, which recruits the E3 ubiquitin ligase Listerin that ubiquitinates NCs. The mechanism of subsequent ribosomal release of Ub-NCs remains obscure. We found that, in non-ubiquitinated 60S RNCs and 80S RNCs formed on non-stop mRNAs, tRNA is not firmly fixed in the P site, which allows peptidyl-tRNA hydrolase Ptrh1 to cleave NC-tRNA, suggesting the existence of a pathway involving release of non-ubiquitinated NCs. Association with NEMF and Listerin and ubiquitination of NCs results in accommodation of NC-tRNA, rendering 60S RNCs resistant to Ptrh1 but susceptible to ANKZF1, which induces specific cleavage in the tRNA acceptor arm, releasing proteasome-degradable Ub-NCs linked to four 3′-terminal tRNA nucleotides. We also found that TCF25, a poorly characterized RQC component, ensures preferential formation of the K48-ubiquitin linkage.
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•NC-tRNAs are not stably fixed in the P site of 60S RNCs and can be cleaved by Ptrh1•Association with NEMF and ubiquitination of NCs accommodates NC-tRNAs in the P site•Accommodation of Ub-NC-tRNAs makes 60S RNCs Ptrh1 resistant but susceptible to ANKZF1•ANKZF1 induces a specific cut in tRNA, releasing Ub-NCs linked to 4 tRNA nucleotides
Stalled ribosomes arising from interrupted translation are dissociated, after which tRNA-linked polypeptides associated with 60S subunits undergo ubiquitination. Subsequent ribosomal release of ubiquitinated polypeptides is mediated by ANKZF1. Kuroha et al. found that ANKZF1 induces specific cleavage in tRNA, releasing proteasome-degradable ubiquitinated polypeptides linked to four 3′-terminal tRNA nucleotides.
The evolutionarily conserved Ski2-Ski3-Ski8 (Ski) complex containing the 3′→5′ RNA helicase Ski2 binds to 80S ribosomes near the mRNA entrance and facilitates 3′→5′ exosomal degradation of mRNA ...during ribosome-associated mRNA surveillance pathways. Here, we assayed Ski’s activity using an in vitro reconstituted translation system and report that this complex efficiently extracts mRNA from 80S ribosomes in the 3′→5′ direction in a nucleotide-by-nucleotide manner. The process is ATP dependent and can occur on pre- and post-translocation ribosomal complexes. The Ski complex can engage productively with mRNA and extract it from 80S complexes containing as few as 19 (but not 13) 3′-terminal mRNA nucleotides starting from the P site. The mRNA-extracting activity of the Ski complex suggests that its role in mRNA quality control pathways is not limited to acceleration of exosomal degradation and could include clearance of stalled ribosomes from mRNA, poising mRNA for degradation and rendering stalled ribosomes recyclable by Pelota/Hbs1/ABCE1.
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•The evolutionarily conserved Ski complex extracts mRNA from 80S ribosomal complexes•mRNA extraction occurs in the 3′→5′ direction in a nucleotide-by-nucleotide manner•The process is ATP dependent and can occur on pre- and post-translocation ribosomes•19 mRNA nucleotides from the P site are sufficient for Ski-mediated mRNA extraction
The evolutionarily conserved Ski complex containing the 3′→5′ RNA helicase Ski2 binds to 80S ribosomes near the mRNA entrance. Zinoviev et al. found that Ski extracts mRNA from 80S ribosomal complexes in the 3′→5′ direction in a nucleotide-by-nucleotide manner. Ski-mediated mRNA extraction renders ribosomal complexes susceptible to recycling by Pelota/Hbs1/ABCE1.
Reinitiation is a strategy used by viruses to express several cistrons from one mRNA. Although extremely weak after translation of long open reading frames (ORFs) on cellular mRNAs, reinitiation ...occurs efficiently on subgenomic bicistronic calicivirus mRNAs, enabling synthesis of minor capsid proteins. The process is governed by a short element upstream of the restart AUG, designated “termination upstream ribosomal binding site” (TURBS). It contains the conserved Motif 1 complementary to h26 of 18S rRNA, displayed in the loop of a hairpin formed by species-specific Motifs 2/2∗. To determine the advantages conferred on reinitiation by TURBS, we reconstituted this process in vitro on two model bicistronic calicivirus mRNAs. We found that post-termination ribosomal tethering of mRNA by TURBS allows reinitiation by post-termination 80S ribosomes and diminishes dependence on eukaryotic initiation factor 3 (eIF3) of reinitiation by recycled 40S subunits, which can be mediated either by eIFs 2/1/1A or by Ligatin following ABCE1-dependent or -independent splitting of post-termination complexes.
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•eIF3 is not essential for reinitiation on calicivirus mRNAs by recycled 40S subunits•Reinitiation on calicivirus mRNAs by 40S subunits requires eIFs 2/1/1A or Ligatin•Reinitiation on calicivirus mRNAs can also be executed by post-termination ribosomes•These modes of calicivirus reinitiation all depend on the integrity of TURBS elements
Reinitiation on calicivirus mRNAs is governed by an element upstream of the restart AUG, which base-pairs with 18S rRNA. Zinoviev et al. found that such tethering allows reinitiation by post-termination ribosomes and diminishes eIF3 dependence of reinitiation by recycled 40S subunits.
Giant viruses have extraordinarily large dsDNA genomes, and exceptionally, they encode various components of the translation apparatus, including tRNAs, aminoacyl-tRNA synthetases and translation ...factors. Here, we focused on the elongation factor 1 (EF1) family of viral translational GTPases (trGTPases), using computational and functional approaches to shed light on their functions. Multiple sequence alignment indicated that these trGTPases clustered into two groups epitomized by members of Mimiviridae and Marseilleviridae, respectively. trGTPases in the first group were more closely related to GTP-binding protein 1 (GTPBP1), whereas trGTPases in the second group were closer to eEF1A, eRF3 and Hbs1. Functional characterization of representative GTPBP1-like trGTPases (encoded by Hirudovirus, Catovirus and Moumouvirus) using in vitro reconstitution revealed that they possess eEF1A-like activity and can deliver cognate aa-tRNAs to the ribosomal A site during translation elongation. By contrast, representative eEF1A/eRF3/Hbs1-like viral trGTPases, encoded by Marseillevirus and Lausannevirus, have eRF3-like termination activity and stimulate peptide release by eRF1. Our analysis identified specific aspects of the functioning of these viral trGTPases with eRF1 of human, amoebal and Marseillevirus origin.
GTP-binding protein 1 (GTPBP1) and GTPBP2 comprise a divergent group of translational GTPases with obscure functions, which are most closely related to eEF1A, eRF3, and Hbs1. Although recent reports ...implicated GTPBPs in mRNA surveillance and ribosome-associated quality control, how they perform these functions remains unknown. Here, we demonstrate that GTPBP1 possesses eEF1A-like elongation activity, delivering cognate aminoacyl-transfer RNA (aa-tRNA) to the ribosomal A site in a GTP-dependent manner. It also stimulates exosomal degradation of mRNAs in elongation complexes. The kinetics of GTPBP1-mediated elongation argues against its functioning in elongation per se but supports involvement in mRNA surveillance. Thus, GTP hydrolysis by GTPBP1 is not followed by rapid peptide bond formation, suggesting that after hydrolysis, GTPBP1 retains aa-tRNA, delaying its accommodation in the A site. In physiological settings, this would cause ribosome stalling, enabling GTPBP1 to elicit quality control programs; e.g., by recruiting the exosome. GTPBP1 can also deliver deacylated tRNA to the A site, indicating that it might function via interaction with deacylated tRNA, which accumulates during stresses. Although GTPBP2's binding to GTP was stimulated by Phe-tRNA
, suggesting that its function might also involve interaction with aa-tRNA, GTPBP2 lacked elongation activity and did not stimulate exosomal degradation, indicating that GTPBP1 and GTPBP2 have different functions.
Protein translation typically begins with the recruitment of the 43S ribosomal complex to the 5′ cap of mRNAs by a cap-binding complex. However, some transcripts are translated in a cap-independent ...manner through poorly understood mechanisms. Here, we show that mRNAs containing N6-methyladenosine (m6A) in their 5′ UTR can be translated in a cap-independent manner. A single 5′ UTR m6A directly binds eukaryotic initiation factor 3 (eIF3), which is sufficient to recruit the 43S complex to initiate translation in the absence of the cap-binding factor eIF4E. Inhibition of adenosine methylation selectively reduces translation of mRNAs containing 5′UTR m6A. Additionally, increased m6A levels in the Hsp70 mRNA regulate its cap-independent translation following heat shock. Notably, we find that diverse cellular stresses induce a transcriptome-wide redistribution of m6A, resulting in increased numbers of mRNAs with 5′ UTR m6A. These data show that 5′ UTR m6A bypasses 5′ cap-binding proteins to promote translation under stresses.
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•m6A residues within the 5′ UTR promote cap-independent translation•Translation of cellular mRNAs is increased by the presence of m6A within the 5′ UTR•Heat shock induces Hsp70 translation in an m6A-dependent manner•Diverse cellular stresses increase 5′ UTR adenosine methylation
N6-methyladenosine (m6A) residues within the 5′ UTR of mRNAs promote translation initiation through a mechanism that does not require the 5′ cap or cap-binding proteins. Diverse cellular stresses selectively increase the levels of m6A within 5′ UTRs, suggesting that 5′ UTR m6A is important for mediating stress-induced translational responses.
Protein translation typically begins with the recruitment of the 43S ribosomal complex to the 5' cap of mRNAs by a cap-binding complex. However, some transcripts are translated in a cap-independent ...manner through poorly understood mechanisms. Here, we show that mRNAs containing N(6)-methyladenosine (m(6)A) in their 5' UTR can be translated in a cap-independent manner. A single 5' UTR m(6)A directly binds eukaryotic initiation factor 3 (eIF3), which is sufficient to recruit the 43S complex to initiate translation in the absence of the cap-binding factor eIF4E. Inhibition of adenosine methylation selectively reduces translation of mRNAs containing 5'UTR m(6)A. Additionally, increased m(6)A levels in the Hsp70 mRNA regulate its cap-independent translation following heat shock. Notably, we find that diverse cellular stresses induce a transcriptome-wide redistribution of m(6)A, resulting in increased numbers of mRNAs with 5' UTR m(6)A. These data show that 5' UTR m(6)A bypasses 5' cap-binding proteins to promote translation under stresses.
In eukaryotes, exposure to stress conditions causes a shift from cap-dependent to cap-independent translation. In trypanosomatids, environmental switches are the driving force of a developmental ...program of gene expression, but it is yet unclear how their translation machinery copes with their constantly changing environment. Trypanosomatids have a unique cap structure (cap-4) and encode four highly diverged paralogs of the cap-binding protein, eIF4E; none were found to genetically complement a yeast mutant failing to express eIF4E. Here we show that in promastigotes, a typical cap-binding complex is anchored through LeishIF4E-4, which associates with components of the cap-binding pre-initiation complex. In axenic amastigotes, expression of LeishIF4E-4 decreases and the protein does not bind the cap, whereas LeishIF4E-1 maintains its expression level and associates with the cap structure and with translation initiation factors. However, LeishIF4E-1 does not interact with eIF4G-like proteins in both life stages, excluding its involvement in cap-dependent translation. Using pull-down assays and mass-spectrometry, we identified a novel, non-conserved 4E-Interacting Protein (Leish4E-IP), which binds to LeishIF4E-1 in promastigotes, but not in amastigotes. Yeast two-hybrid and NMR spectroscopy confirmed the specificity of this interaction. We propose that Leish4E-IP is a translation regulator that is involved in switching between cap-dependent and alternative translation pathways.
Ribosomal stalling induces the ribosome-associated quality control (RQC) pathway targeting aberrant polypeptides. RQC is initiated by K63-polyubiquitination of ribosomal protein uS10 located at the ...mRNA entrance of stalled ribosomes by the E3 ubiquitin ligase ZNF598 (Hel2 in yeast). Ubiquitinated ribosomes are dissociated by the ASC-1 complex (ASCC) (RQC-Trigger (RQT) complex in yeast). A cryo-EM structure of the ribosome-bound RQT complex suggested the dissociation mechanism, in which the RNA helicase Slh1 subunit of RQT (ASCC3 in mammals) applies a pulling force on the mRNA, inducing destabilizing conformational changes in the 40S subunit, whereas the collided ribosome acts as a wedge, promoting subunit dissociation. Here, using an in vitro reconstitution approach, we found that ribosomal collision is not a strict prerequisite for ribosomal ubiquitination by ZNF598 or for ASCC-mediated ribosome release. Following ubiquitination by ZNF598, ASCC efficiently dissociated all polysomal ribosomes in a stalled queue, monosomes assembled in RRL, in vitro reconstituted 80S elongation complexes in pre- and post-translocated states, and 48S initiation complexes, as long as such complexes contained ≥ 30-35 3'-terminal mRNA nt. downstream from the P site and sufficiently long ubiquitin chains. Dissociation of polysomes and monosomes both involved ribosomal splitting, enabling Listerin-mediated ubiquitination of 60S-associated nascent chains.
Abstract
Leishmania parasites are unicellular pathogens that are transmitted to humans through the bite of infected sandflies. Most of the regulation of their gene expression occurs ...post-transcriptionally, and the different patterns of gene expression required throughout the parasites' life cycle are regulated at the level of translation. Here, we report the X-ray crystal structure of the Leishmania cap-binding isoform 1, LeishIF4E-1, bound to a protein fragment of previously unknown function, Leish4E-IP1, that binds tightly to LeishIF4E-1. The molecular structure, coupled to NMR spectroscopy experiments and in vitro cap-binding assays, reveal that Leish4E-IP1 allosterically destabilizes the binding of LeishIF4E-1 to the 5′ mRNA cap. We propose mechanisms through which Leish4E-IP1-mediated LeishIF4E-1 inhibition could regulate translation initiation in the human parasite.
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