Canonical Eukaryotic Initiation Factors Determine Initiation of Translation by Internal Ribosomal Entry

Abstract

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...

Bunyavirus N: eIF4F surrogate and cap-guardian

“…In many of the picornaviruses, such as encephalomyocarditis virus, localization of the 43S pre-initiation complex requires the eIF4G and eIF4A components of eIF4F. [14][15][16] However, in the case of the flavivirus, hepatitis C virus (HCV), as well as some of the avian and porcine picornaviruses, the pre-initiation complex is recruited directly to the IRES by way of just the 40S ribosomal subunit and eIF3, and without eIF4F components. [17][18][19] Further, the dicistoviruses, such as cricket paralysis virus, recruit the 40S subunit its IRES without either eIF4F or eIF3.…”

A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initation of hepatitis C and classical swine fever virus RNAs

“…The canonical factors eIF2, eIF3, eIF4A, eIF4B, and eIF4F mediate attachment (''internal entry'') of 40S ribosomal subunits to the EMCV IRES (Pestova et al 1996a). Purified 40S subunits also bound the HCV IRES in the presence of Met-tRNA i Met , ATP, GMP-PNP, and these five factors ( Fig.…”

“…IRES-mediated initiation is used by several cellular mRNAs that encode regulatory proteins, and has been usurped by numerous viruses (e.g., Pestova et al 1996a). Two major groups of viral IRESs have been iden-tified; there is no obvious similarity between them or other viral or cellular IRESs.…”

A gastrin transcript expressed in gastrointestinal cancer cells contains an internal ribosome entry site

“…They appear to include canonical translation initiation factors such as eIF2, eIF3 and eIF4F (Pestova et al, 1996) and also other trans-acting factors, and may act to change the secondary structure of the IRES to facilitate ribosomal binding Pickering et al, 2004).…”