PREFACE Protein synthesis is principally regulated at the initiation stage (rather than during elongation or termination), allowing rapid, reversible and spatial control over gene expression. Progress over recent years in determining the structures and activities of initiation factors, and in mapping their interactions within ribosomal initiation complexes, has significantly advanced our understanding of the complex translation initiation process. These developments have provided a solid foundation for studies of regulation of initiation by mechanisms that include modulation of the activity of initiation factors (which affects almost all scanning-dependent initiation), or via sequence-specific RNA-binding proteins and microRNAs (which thus impact individual mRNAs).

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.

Initiation of translation of hepatitis C virus and classical swine fever virus mRNAs results from internal ribosomal entry. We reconstituted internal ribosomal entry in vitro from purified translation components and monitored assembly of 48S ribosomal preinitiation complexes by toe-printing. Ribosomal subunits (40S) formed stable binary complexes on both mRNAs. The complex structure of these RNAs determined the correct positioning of the initiation codon in the ribosomal ''P'' site in binary complexes. Ribosomal binding and positioning on these mRNAs did not require the initiation factors eIF3, eIF4A, eIF4B, and eIF4F and translation of these mRNAs was not inhibited by a trans-dominant eIF4A mutant. Addition of Met-tRNA i Met , eIF2, and GTP to these binary ribosomal complexes resulted in formation of 48S preinitiation complexes. The striking similarities between this eukaryotic initiation mechanism and the mechanism of translation initiation in prokaryotes are discussed. Protein synthesis begins following assembly of an initiation complex in which the initiation codon of an mRNA and the anticodon of initiator tRNA are base-paired in the ribosomal ''P'' site. There are similarities and significant differences in the mechanisms of initiation complex formation in prokaryotes and eukaryotes. A universal characteristic is that initiation starts with separated ribosomal subunits.In prokaryotes, the small (30S) ribosomal subunit binds mRNA and initiator tRNA in random order to form a complex that then undergoes conformational rearrangement, promoting codon-anticodon base-pairing at the P site and joining of the large (50S) subunit (Gualerzi and Pon 1990). Ribosome binding results from interactions between the 30S subunit and multiple recognition elements in the mRNA, such as the Shine-Dalgarno sequence (McCarthy and Brimacombe 1994). It does not depend on initiation factors. Ribosome-binding sites can occur at any position within an mRNA and as a result many prokaryotic mRNAs are polycistronic.In contrast, the small (40S) ribosomal subunit in eukaryotes requires several eukaryotic initiation factors (eIFs), first to bind initiator tRNA (as a ternary complex with eIF2 and GTP) to form a 43S complex and then to bind mRNA to form a 48S complex (Merrick 1992). The most common mechanism for recruitment of an mRNA is mediated by its capped 5Ј end, which is bound by the eIF4E subunit of eIF4F and then by the 43S complex. Ribosomal binding to mRNA and scanning to the initiation codon require ATP hydrolysis and involve eIF4A, eIF4B, and eIF4F. Most mRNAs that use this mechanism of ribosomal binding are monocistronic because initiation is usually limited to the 5Ј-most AUG codon.

To elucidate an outline of the mechanism of eukaryotic translation initiation, 48S complex formation was analyzed on defined mRNAs in reactions reconstituted in vitro from fully purified translation components. We found that a ribosomal 40S subunit, eukaryotic initiation factor (eIF) 3, and the eIF2 ternary complex form a 43S complex that can bind to the 5-end of an unstructured 5-untranslated region (5-UTR) and in the presence of eIF1 scan along it and locate the initiation codon without a requirement for adenosine triphosphate (ATP) or factors (eIF4A, eIF4B, eIF4F) associated with ATP hydrolysis. Scanning on unstructured 5-UTRs was enhanced by ATP, eIFs 4A and 4B, and the central domain of the eIF4G subunit of eIF4F. Their omission increased the dependence of scanning on eIFs 1 and 1A. Ribosomal movement on 5-UTRs containing even weak secondary structures required ATP and RNA helicases. eIF4F was essential for scanning, and eIFs 4A and 4B were insufficient to promote this process in the absence of eIF4F. We report that in addition to its function in scanning, eIF1 also plays a principal role in initiation codon selection. In the absence of eIF1, 43S complexes could no longer discriminate between cognate and noncognate initiation codons or sense the nucleotide context of initiation codons and were able to assemble 48S complexes on 5-proximal AUG triplets located only 1, 2, and 4 nt from the 5-end of mRNA.

Translation initiation is a complex process in which initiator tRNA, 40S, and 60S ribosomal subunits are assembled by eukaryotic initiation factors (eIFs) into an 80S ribosome at the initiation codon of mRNA. The cap-binding complex eIF4F and the factors eIF4A and eIF4B are required for binding of 43S complexes (comprising a 40S subunit, eIF2͞GTP͞Met-tRNAi and eIF3) to the 5 end of capped mRNA but are not sufficient to promote ribosomal scanning to the initiation codon. eIF1A enhances the ability of eIF1 to dissociate aberrantly assembled complexes from mRNA, and these factors synergistically mediate 48S complex assembly at the initiation codon. Joining of 48S complexes to 60S subunits to form 80S ribosomes requires eIF5B, which has an essential ribosome-dependent GTPase activity and hydrolysis of eIF2-bound GTP induced by eIF5. Initiation on a few mRNAs is cap-independent and occurs instead by internal ribosomal entry. Encephalomyocarditis virus (EMCV) and hepatitis C virus epitomize distinct mechanisms of internal ribosomal entry site (IRES)-mediated initiation. The eIF4A and eIF4G subunits of eIF4F bind immediately upstream of the EMCV initiation codon and promote binding of 43S complexes. EMCV initiation does not involve scanning and does not require eIF1, eIF1A, and the eIF4E subunit of eIF4F. Initiation on some EMCV-like IRESs requires additional noncanonical initiation factors, which alter IRES conformation and promote binding of eIF4A͞4G. Initiation on the hepatitis C virus IRES is even simpler: 43S complexes containing only eIF2 and eIF3 bind directly to the initiation codon as a result of specific interaction of the IRES and the 40S subunit.T ranslation of mRNA into protein begins after assembly of initiator tRNA (Met-tRNA i ), mRNA, and separated 40S and 60S ribosomal subunits into an 80S ribosome in which MettRNA i is positioned in the ribosomal P site at the initiation codon. The complex initiation process that leads to 80S ribosome formation consists of several linked stages that are mediated by eukaryotic initiation factors. These stages are:(i) Selection of initiator tRNA from the pool of elongator tRNAs by eukaryotic initiation factor (eIF)2 and binding of an eIF2͞GTP͞Met-tRNA i ternary complex and other eIFs to the 40S subunit to form a 43S preinitiation complex.(ii) Binding of the 43S complex to mRNA, which in most instances occurs by a mechanism that involves initial recognition of the m 7 G cap at the mRNA 5Ј-terminus by the eIF4E (cap-binding) subunit of eIF4F. Ribosomes bind to a subset of cellular and viral mRNAs as a result of cap-and endindependent internal ribosomal entry.(iii) Movement of the mRNA-bound ribosomal complex along the 5Ј nontranslated region (5ЈNTR) from its initial binding site to the initiation codon to form a 48S initiation complex in which the initiation codon is base paired to the anticodon of initiator tRNA.(iv) Displacement of factors from the 48S complex and joining of the 60S subunit to form an 80S ribosome, leaving Met-tRNA i in the ribosomal P site.Research in ...

Translation of picornavirus RNA is initiated after ribosomal binding to an internal ribosomal entry site (IRES) within the 5 untranslated region. We have reconstituted IRES-mediated initiation on encephalomyocarditis virus RNA from purified components and used primer extension analysis to confirm the fidelity of 48S preinitiation complex formation. Eukaryotic initiation factor 2 (eIF2), eIF3, and eIF4F were required for initiation; eIF4B and to a lesser extent the pyrimidine tract-binding protein stimulated this process. We show that eIF4F binds to the IRES in a novel cap-independent manner and suggest that cap-and IRES-dependent initiation mechanisms utilize different modes of interaction with this factor to promote ribosomal attachment to mRNA.Initiation of eukaryotic protein synthesis is the process of assembly of an 80S initiation complex containing initiator tRNA i Met , 40S, and 60S ribosomal subunits at the initiation codon of an mRNA (42). The first step in this process is formation of a 43S complex that consists of eukaryotic initiation factor 2 (eIF2), eIF3, and initiator Met-tRNA i Met bound to the 40S subunit. The second, rate-limiting step is the binding of mRNA to the 43S complex to form a 48S preinitiation complex. This occurs in one of two ways.The first mechanism is characteristic of most mRNAs. It involves recognition of the m 7 GpppX cap at the free 5Ј end of mRNA by the 4E subunit of eIF4F, followed by binding of the 43S complex at or close to the cap. Binding and 5Ј-3Ј scanning by this complex to the first AUG codon require ATP hydrolysis and unwinding of secondary structure in the 5Ј untranslated region (UTR) by eIF4F or eIF4A in cooperation with eIF4B. This mechanism of ribosomal entry imposes several restrictions on the structure of mRNAs that use it: they are capped, have relatively short, unstructured 5Ј UTRs, and are monocistronic because initiation is limited to the 5Ј-most AUG codon (34).A second, cap-independent mechanism of ribosome binding is used by mRNAs that contain an internal ribosomal entry site (IRES). IRES-mediated initiation is used by a number of cellular mRNAs, including those that encode the transcription factors TFIID (TATA-binding protein) and HAP4 (25), the growth factors fibroblast growth factor 2 and insulin-like growth factor 2 (63, 67), the homeotic genes Antennapedia and Ultrabithorax (45), the translation initiation factor eIF4G (14), and the immunoglobulin heavy-chain binding protein (39). This mechanism of translation initiation has been usurped by a number of viruses (e.g., references 49 and 65) and is exemplified by encephalomyocarditis virus (EMCV) (21, 26). The EMCV IRES is about 450 nucleotides (nt) long, is highly structured, and lies immediately upstream of AUG 834 , which is the 11th AUG triplet in the 5Ј UTR and the initiation codon for synthesis of the viral polyprotein (11, 27, 52, 66). The 5Ј UTRs of IRES-containing mRNAs differ from those of conventional mRNAs in many respects: they contain multiple AUG triplets and extensive secondary structure...

Positioning of the translation initiation complex on mRNAs requires interaction between the anticodon of initiator Met-tRNA, associated with eIF2-GTP and 40S ribosomal subunit, and the cognate start codon of the mRNA. We show that an internal ribosome entry site located in the genome of cricket paralysis virus can form 80S ribosomes without initiator Met-tRNA, eIF2, or GTP hydrolysis, with a CCU triplet in the ribosomal P site and a GCU triplet in the A site. P-site mutagenesis revealed that the P site was not decoded, and protein sequence analysis showed that translation initiates at the triplet in the A site. Translational initiation from the A site of the ribosome suggests that the repertoire of translated open reading frames in eukaryotic mRNAs may be greater than anticipated.

The scanning model of translation initiation is a coherent description of how eukaryotic ribosomes reach the initiation codon after being recruited to the capped 5' end of messenger RNA. Five eukaryotic initiation factors (eIF 2, 3, 4A, 4B and 4F) with established functions have been assumed to be sufficient to mediate this process. Here we report that eIF1 and eIF1A are also both essential for translation initiation. In their absence, 43S ribosomal preinitiation complexes incubated with ATP, eIF4A, eIF4B and eIF4F bind exclusively to the cap-proximal region but are unable to reach the initiation codon. Individually, eIF1A enhances formation of this cap-proximal complex, and eIF1 weakly promotes formation of a 48S ribosomal complex at the initiation codon. These proteins act synergistically to mediate assembly of ribosomal initiation complexes at the initiation codon and dissociate aberrant complexes from the mRNA.