SUMMARY 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.

After termination, eukaryotic 80S ribosomes remain associated with mRNA, P-site deacylated tRNA and release factor eRF1, and must be recycled by dissociating these ligands and separating ribosomes into subunits. Although recycling of eukaryotic post-termination complexes (post-TCs) can be mediated by initiation factors eIF3, eIF1 and eIF1A (Pisarev et al., 2007), this energy-free mechanism can function only in a narrow range of low Mg2+ concentrations. Here we report that ABCE1, a conserved and essential member of the ATP-binding cassette (ABC) family of proteins, promotes eukaryotic ribosomal recycling over a wide range of Mg2+ concentrations. ABCE1 dissociates post-TCs into free 60S subunits and mRNA- and tRNA-bound 40S subunits. It can hydrolyze ATP, GTP, UTP and CTP. NTP hydrolysis by ABCE1 is stimulated by post-TCs and is required for its recycling activity. Importantly, ABCE1 dissociates only post-TCs obtained with eRF1/eRF3 (or eRF1 alone), but not post-TCs obtained with puromycin in eRF1's absence.

No-go decay (NGD) and non-stop decay (NSD) are eukaryotic surveillance mechanisms that target mRNAs on which elongation complexes (ECs) are stalled by, for example, stable secondary structures (NGD) or due to the absence of a stop codon (NSD). Two interacting proteins Dom34(yeast)/Pelota(mammals) and Hbs1, which are paralogues of eRF1 and eRF3, are implicated in these processes. Dom34/Hbs1 were shown to promote dissociation of stalled ECs and release of intact peptidyl-tRNA. Using an in vitro reconstitution approach, we investigated the activities of mammalian Pelota/Hbs1 and report that Pelota/Hbs1 also induced dissociation of ECs and release of peptidyl-tRNA, but only in the presence of ABCE1. Whereas Pelota and ABCE1 were essential, Hbs1 had a stimulatory effect. Importantly, ABCE1/Pelota/Hbs1 dissociated ECs containing only a limited number of mRNA nucleotides downstream of the P-site, which suggests that ABCE1/Pelota/Hbs1 would disassemble NSD complexes stalled at the 3 0 -end, but not pre-cleavage NGD complexes stalled in the middle of mRNA. ABCE1/Pelota/Hbs1 also dissociated vacant 80S ribosomes, which stimulated 48S complex formation, suggesting that Pelota/Hbs1 have an additional role outside of NGD.

Met i ) by eukaryotic initiation factor eIF2. In cooperation with eIF3, eIF1, and eIF1A, Met-tRNA Met i /eIF2/ GTP binds to 40S subunits yielding 43S preinitiation complexes that attach to the 59-terminal region of mRNAs and then scan to the initiation codon to form 48S initiation complexes with established codon-anticodon basepairing. Stress-activated phosphorylation of eIF2a reduces the level of active eIF2, globally inhibiting translation. However, translation of several viral mRNAs, including Sindbis virus (SV) 26S mRNA and mRNAs containing hepatitis C virus (HCV)-like IRESs, is wholly or partially resistant to inhibition by eIF2 phosphorylation, despite requiring Met-tRNA Met i . Here we report the identification of related proteins that individually (Ligatin) or together (the oncogene MCT-1 and DENR, which are homologous to N-terminal and C-terminal regions of Ligatin, respectively) promote efficient eIF2-independent recruitment of Met-tRNA Met i to 40S/mRNA complexes, if attachment of 40S subunits to the mRNA places the initiation codon directly in the P site, as on HCV-like IRESs and, as we show here, SV 26S mRNA. In addition to their role in initiation, Ligatin and MCT-1/DENR can promote release of deacylated tRNA and mRNA from recycled 40S subunits after ABCE1-mediated dissociation of post-termination ribosomes. During the first stage of protein synthesis, initiation, the P site of the small ribosomal subunit is occupied by aminoacylated initiator tRNA (Met-tRNA Met i ), whereas, at the last stage, ribosomal recycling, it contains deacylated elongator tRNA, which remains bound to the ribosome after the hydrolysis of peptidyl-tRNA and release of peptide that occur during termination. In both bacteria and eukaryotes, occupancy of the P site during initiation and ribosomal recycling is regulated by initiation factors. However, the mechanisms of ribosomal attachment of Met-tRNA Met i during initiation, and the factors involved in this process, differ considerably between the two kingdoms. In bacteria, 30S ribosomal subunits bind to mRNA directly via the Shine-Dalgarno (SD) interaction, which places the initiation codon into the 30S subunit's P site. fMet-tRNA fMet i binds to 30S subunits that are associated with all three initiation factors (IF1, IF2, and IF3) and (usually) mRNA, and the ribosome-bound IF2 accelerates its attachment by either direct interaction with the

Ribosomal recruitment of cellular mRNAs depends on binding of eIF4F to the mRNA’s 5′-terminal ‘cap’. The minimal ‘cap0’ consists of N7-methylguanosine linked to the first nucleotide via a 5′-5′ triphosphate (ppp) bridge. Cap0 is further modified by 2′-O-methylation of the next two riboses, yielding ‘cap1’ (m7GpppNmN) and ‘cap2’ (m7GpppNmNm). However, some viral RNAs lack 2′-O-methylation, whereas others contain only ppp- at their 5′-end. Interferon-induced proteins with tetratricopeptide repeats (IFITs) are highly expressed effectors of innate immunity that inhibit viral replication by incompletely understood mechanisms. Here, we investigated the ability of IFIT family members to interact with cap1-, cap0- and 5′ppp- mRNAs and inhibit their translation. IFIT1 and IFIT1B showed very high affinity to cap-proximal regions of cap0-mRNAs (K1/2,app ∼9 to 23 nM). The 2′-O-methylation abrogated IFIT1/mRNA interaction, whereas IFIT1B retained the ability to bind cap1-mRNA, albeit with reduced affinity (K1/2,app ∼450 nM). The 5′-terminal regions of 5′ppp-mRNAs were recognized by IFIT5 (K1/2,app ∼400 nM). The activity of individual IFITs in inhibiting initiation on a specific mRNA was determined by their ability to interact with its 5′-terminal region: IFIT1 and IFIT1B efficiently outcompeted eIF4F and abrogated initiation on cap0-mRNAs, whereas inhibition on cap1- and 5′ppp- mRNAs by IFIT1B and IFIT5 was weaker and required higher protein concentrations.

During ribosome recycling, post-termination complexes are dissociated by ABCE1 and eRF1 into 60S and tRNA/mRNA-associated 40S subunits, after which tRNA/mRNA are released by eIF1/eIF1A, Ligatin or MCT1/DENR. In some instances, 40S subunits remain associated with mRNA and reinitiate at nearby AUGs. Here, we recapitulated reinitiation in vitro using a reconstituted mammalian translation system. The presence of eIFs 2/3/1/1A and Met-tRNAiMet was sufficient for recycled 40S subunits to remain on mRNA, scan bidirectionally and reinitiate both at upstream and downstream AUGs if mRNA regions flanking the stop codon were unstructured. Imposition of 3’-directionality additionally required eIF4F. Strikingly, post-termination 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>eRF1/eRF3), the nature of tRNA, and was enhanced by eEF2. The mobility of post-termination ribosomes suggests that some reinitiation events could involve 80S ribosomes rather than 40S subunits.

YB-1 is a DNA/RNA-binding nucleocytoplasmic shuttling protein whose regulatory effect on many DNA-and RNAdependent events is determined by its localization in the cell. Distribution of YB-1 between the nucleus and the cytoplasm is known to be dependent on nuclear targeting and cytoplasmic retention signals located within the C-terminal portion of YB-1. Here, we report that YB-1 undergoes a specific proteolytic cleavage by the 20S proteasome, which splits off the C-terminal 105-amino-acidlong YB-1 fragment containing a cytoplasmic retention signal. Cleavage of YB-1 by the 20S proteasome in vitro appears to be ubiquitin-and ATP-independent, and is abolished by the association of YB-1 with messenger RNA. We also found that genotoxic stress triggers a proteasome-mediated cleavage of YB-1 in vivo and leads to accumulation of the truncated protein in nuclei of stressed cells. Endoproteolytic activity of the proteasome may therefore play an important role in regulating YB-1 functioning, especially under certain stress conditions.

Summary The Cricket paralysis virus (CrPV) uses an internal ribosomal entry site (IRES) to hijack the ribosome. In a remarkable RNA-based mechanism involving neither initiation factor nor initiator tRNA, the CrPV IRES jump starts translation in the elongation phase from the ribosomal A-site. Here we present cryo-EM maps of 80S•CrPV-STOP•eRF1•eRF3•GMPPNP and 80S•CrPV-STOP•eRF1 complexes revealing a previously unseen binding state of the IRES and directly rationalizing that an eEF2-dependent translocation of the IRES is required to allow the first A-site occupation. During this unusual translocation event the IRES undergoes a pronounced conformational change to a more stretched conformation. At the same time our structural analysis provides information about the binding modes of eRF1•eRF3•GMPPNP and eRF1 in a minimal system. It shows that neither eRF3 nor ABCE1 are required for the active conformation of eRF1 at the intersection between eukaryotic termination and recycling.