Conserved nucleotides within the J domain of the encephalomyocarditis virus internal ribosome entry site are required for activity and for interaction with eIF4G.
The internal ribosome entry site (IRES) elements of cardioviruses (e.g., encephalomyocarditis virus [EMCV] and foot-and-mouth disease virus) are predicted to have very similar secondary structures. Among these complex RNA structures there is only rather limited complete sequence conservation. Within the J domain of the EMCV IRES there are four highly conserved nucleotides (A704, C705, G723, and A724)., which are predicted to be unpaired and have been targeted for mutagenesis. Using an IRES-dependent cell selection system, we have isolated functional IRES elements from a pool of up to 256 mutants. All changes to these conserved nucleotides resulted in IRES elements that were less efficient at directing internal initiation of translation than the wild-type element, and even some of the single point mutants were highly defective. Each of the mutations adversely affected the ability of the RNAs to interact with the translation initiation factor eIF4G.
Clark
Angela T
AT
BBSRC Institute for Animal Health, Pirbright, Woking, Surrey GU24 ONF, United Kingdom.
Robertson
Morwenna E M
ME
Conn
Graeme L
GL
Belsham
Graham J
GJ
D000818:Animals; D001483:Base Sequence; D002478:Cells, Cultured; D017124:Conserved Sequence; D006224:Cricetinae; D017931:DNA Primers; D004686:Encephalomyocarditis virus / Q000235:genetics* / Q000378:metabolism; D039603:Eukaryotic Initiation Factor-4G / Q000378:metabolism*; D016296:Mutagenesis; D011485:Protein Binding; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012270:Ribosomes / Q000378:metabolism*
D017931:DNA Primers; D039603:Eukaryotic Initiation Factor-4G