We are interested in the mechanism of initiation site selection on eukaryotic mRNAs, and the ways in which viruses have subverted this mechanism. Of all the steps in protein synthesis, initiation is the one which differs most radically between eukaryotes and prokaryotes, requiring no less than 11 separate initiation factor proteins (a total of 26 polypeptides of aggregate mass >1600 kDa) in eukaryotes as against 3 such factors, each a single polypeptide chain, in eubacteria.
The initiation site in almost all eukaryotic cellular mRNAs is the 5’-proximal AUG triplet. This is accessed by a process in which the small ribosomal subunit scans the mRNA from the capped end, stopping usually at the first AUG (subject to some influence of local sequence context). If the first AUG is followed by a short open-reading frame (<~20 codons), some ribosomes may resume scanning and re-initiate translation at a downstream AUG. Our current and recent interests include the ‘mechanics’ of the scanning process, and the reason why reinitiation usually occurs only if the first cistron is short.
In some cases, initiation is by direct ribosome entry at an internal site in the mRNA, dependent on a substantial (up to 450 nt.) cis-acting motif in the RNA, known as an IRES (for ‘internal ribosome entry segment’), which is generally highly structured. IRESs are identified by the fact that when inserted as the intercistronic spacer of a dicistronic mRNA they promote translation of the downstream cistron (see figure). Three entirely distinct types of IRES have been identified amongst different RNA virus genera: (i) animal picornaviruses; (ii) hepatitis C virus and pestiviruses; and (iii) the dicistrovirus family of insect viruses. The questions concerning internal initiation on each type of IRES that we have addressed and are continuing to examine, include: (a) where is the actual ribosome entry site; (b) which canonical initiation factors and what additional cellular proteins are needed for initiation; and (c) how fundamentally different are the scanning and IRES-dependent mechanisms of initiation?
1. Jackson RJ (2013) The current status of vertebrate cellular mRNA IRESs. Cold Spring Harb Perspect Biol 5(2)
2. Pöyry TA, Kaminski A, Connell EJ, Fraser CS, Jackson RJ (2007) The mechanism of an exceptional case of reinitiation after translation of a long ORF reveals why such events do not generally occur in mammalian mRNA translation. Genes Dev 21(23): 3149-3162
3. Kafasla P, Morgner N, Poyry TA, Curry S, Robinson CV, Jackson RJ (2009) Polypyrimidine tract binding protein stabilizes the encephalomyocarditis virus IRES structure via binding multiple sites in a unique orientation. Mol Cell 34(5): 556-568
4. Kafasla P, Morgner N, Robinson CV, Jackson RJ (2010) Polypyrimidine tract-binding protein stimulates the poliovirus IRES by modulating eIF4G binding. EMBO J 29(21): 3710-3722
5. Poyry TA, Jackson RJ (2011) Mechanisms governing the selection of translation initiation sites on foot-and-mouth disease virus RNA. J Virol 85(19): 10178-10188