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-tRNAiMet 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.
•eIF2, eIF3, eIF1, and eIF1A promote bidirectional reinitiation by recycled 40S subunits•Imposition of 3′ directionality on reinitiation additionally requires eIF4F•Posttermination 80S ribosomes are mobile and can migrate to cognate codons•eEF2 induces dissociation of eRF1 and promotes 80S ribosomal migration
During eukaryotic translation initiation, 43S complexes, comprising a 40S ribosomal subunit, initiator transfer RNA and initiation factors (eIF) 2, 3, 1 and 1A, attach to the 5'-terminal region of ...messenger RNA and scan along it to the initiation codon. Scanning on structured mRNAs also requires the DExH-box protein DHX29. Mammalian eIF3 contains 13 subunits and participates in nearly all steps of translation initiation. Eight subunits having PCI (proteasome, COP9 signalosome, eIF3) or MPN (Mpr1, Pad1, amino-terminal) domains constitute the structural core of eIF3, to which five peripheral subunits are flexibly linked. Here we present a cryo-electron microscopy structure of eIF3 in the context of the DHX29-bound 43S complex, showing the PCI/MPN core at ∼6 Å resolution. It reveals the organization of the individual subunits and their interactions with components of the 43S complex. We were able to build near-complete polyalanine-level models of the eIF3 PCI/MPN core and of two peripheral subunits. The implications for understanding mRNA ribosomal attachment and scanning are discussed.
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.
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.
Eukaryotic translation initiation begins with assembly of a 43S preinitiation complex. First, methionylated initiator methionine transfer RNA (Met-tRNAiMet), eukaryotic initiation factor (eIF) 2, and ...guanosine triphosphate form a ternary complex (TC). The TC, eIF3, eIF1, and eIF1A cooperatively bind to the 40S subunit, yielding the 43S preinitiation complex, which is ready to attach to messenger RNA (mRNA) and start scanning to the initiation codon. Scanning on structured mRNAs additionally requires DHX29, a DExH-box protein that also binds directly to the 40S subunit. Here, we present a cryo-electron microscopy structure of the mammalian DHX29-bound 43S complex at 11.6 Å resolution. It reveals that eIF2 interacts with the 40S subunit via its α subunit and supports Met-tRNAiMet in an unexpected P/I orientation (eP/I). The structural core of eIF3 resides on the back of the 40S subunit, establishing two principal points of contact, whereas DHX29 binds around helix 16. The structure provides insights into eukaryote-specific aspects of translation, including the mechanism of action of DHX29.
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•The eIF3e core binds the 40S subunit via ribosomal proteins S1e, S13e, S26e, and S27e•eIF2 binds the 40S subunit via interaction of the eIF2a-D1 domain with ribosomal protein S5e•Initiator transfer RNA adopts an unexpected P/I orientation close to that in prokaryotic initiation•In DHX29-43S complexes, DHX29 binds h16 of 18S ribosomal RNA, and the mRNA latch is closed
The DHX29 helicase promotes mRNA scanning of the 40S ribosome for translation initiation, and a new structure provides insights into eukaryote-specific aspects of initiation, including how initiator transfer RNA, eIF2, eIF3, and DHX29 interact with the 40S subunit.
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
Translation initiation on structured mammalian mRNAs requires DHX29, a DExH protein that comprises a unique 534-aa-long N-terminal region (NTR) and a common catalytic DExH core. DHX29 binds ...to 40S subunits and possesses 40S-stimulated NTPase activity essential for its function. In the cryo-EM structure of DHX29-bound 43S preinitiation complexes, the main DHX29 density resides around the tip of helix 16 of 18S rRNA, from which it extends through a linker to the subunit interface forming an intersubunit domain next to the eIF1A binding site. Although a DExH core model can be fitted to the main density, the correlation between the remaining density and the NTR is unknown. Here, we present a model of 40S-bound DHX29, supported by directed hydroxyl radical cleavage data, showing that the intersubunit domain comprises a dsRNA-binding domain (dsRBD, aa 377–448) whereas linker corresponds to the long α-helix (aa 460–512) that follows the dsRBD. We also demonstrate that the N-terminal α-helix and the following UBA-like domain form a four-helix bundle (aa 90–166) that constitutes a previously unassigned section of the main density and resides between DHX29’s C-terminal α-helix and the linker. In vitro reconstitution experiments revealed the critical and specific roles of these NTR elements for DHX29’s function.
Ribosomal attachment to mammalian capped mRNAs is achieved through the cap-eukaryotic initiation factor 4E (eIF4E)-eIF4G-eIF3-40S chain of interactions, but the mechanism by which mRNA enters the ...mRNA-binding channel of the 40S subunit remains unknown. To investigate this process, we recapitulated initiation on capped mRNAs in vitro using a reconstituted translation system. Formation of initiation complexes at 5'-terminal AUGs was stimulated by the eIF4E-cap interaction and followed "the first AUG" rule, indicating that it did not occur by backward scanning. Initiation complexes formed even at the very 5' end of mRNA, implying that Met-tRNAi (Met) inspects mRNA from the first nucleotide and that initiation does not have a "blind spot." In assembled initiation complexes, the cap was no longer associated with eIF4E. Omission of eIF4A or disruption of eIF4E-eIF4G-eIF3 interactions converted eIF4E into a specific inhibitor of initiation on capped mRNAs. Taken together, these results are consistent with the model in which eIF4E-eIF4G-eIF3-40S interactions place eIF4E at the leading edge of the 40S subunit, and mRNA is threaded into the mRNA-binding channel such that Met-tRNAi (Met) can inspect it from the first nucleotide. Before entering, eIF4E likely dissociates from the cap to overcome steric hindrance. We also found that the m(7)G cap specifically interacts with eIF3l.
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.
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.
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