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.

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.

The major protein of cytoplasmic mRNPs from rabbit reticulocytes, YB-1, is a member of an ancient family of proteins containing a common structural feature, coldshock domain. In eukaryotes, this family is represented by multifunctional mRNA/Y-box DNA-binding proteins that control gene expression at different stages. To address possible post-transcriptional regulation of YB-1 gene expression, we examined effects of exogenous 5-and 3-untranslatable region-containing fragments of YB-1 mRNA on its translation and stability in a cell-free system. The addition of the 3 mRNA fragment as well as its subfragment I shut off protein synthesis at the initiation stage without affecting mRNA stability. UV crosslinking revealed four proteins (69, 50, 46, and 44 kDa) that specifically interacted with the 3 mRNA fragment; the inhibitory subfragment I bound two of them, 69-and 50-kDa proteins. We have identified these proteins as PABP (poly(A)-binding protein) (69 kDa) and YB-1 (50 kDa) and demonstrated that titrating out of PABP by poly(A) strongly and specifically inhibits YB-1 mRNA cap ؉ poly(A) ؊ translation in a cell-free system. Thus, PABP is capable of positively affecting YB-1 mRNA translation in a poly(A) tail-independent manner.The evolutionarily conserved family of cold-shock domaincontaining proteins (CSD proteins) 1 is represented in organisms from bacteria to man by multifunctional DNA/RNA-binding proteins (1, 2). In bacteria, some of them known as major cold-shock proteins are responsible for adaptation to growth at low temperatures, and their expression is enormously activated with a temperature decrease (3, 4). In mammalian cells, CSD proteins regulate cell proliferation and differentiation and are involved in cell defense systems (5-11). In the cell nucleus, CSD proteins regulate transcription by interacting with promoters and enhancers of many genes (9, 12-17). They are also involved in DNA replication and repair, as well as in mRNA splicing (5, 18 -20). In the cytoplasm, CSD proteins bind mRNAs, affecting their translation fate (21-28) and extending their lifetime (29).In bacteria, accumulation of major cold-shock proteins at low temperatures was shown to result mainly from strong and selective stabilization of their mRNAs (30, 31). The crucial role of eukaryotic CSD proteins in major cellular events suggests that there is precise regulation of their expression. At present the post-transcriptional control of eukaryotic gene expression is to a large extent attributed to the presence of specific sequences within mRNA 5Ј-and 3Ј-untranslatable regions (UTRs), which serve as targets for binding of certain proteins and complementary RNAs (32, 33). Here, we studied this type of regulation during in vitro synthesis of rabbit p50, a member of the eukaryotic CSD protein family, which is the major protein of reticulocyte mRNPs and is virtually identical to the human Y-box binding protein YB-1. To determine a possible role of YB-1 mRNA UTRs in YB-1 post-transcriptional regulation, we examined effects of exogenous mRNA f...