A selection system for functional internal ribosome entry site (IRES) elements: analysis of the requirement for a conserved GNRA tetraloop in the encephalomyocarditis virus IRES.
Picornavirus internal ribosome entry site (IRES) elements direct cap-independent internal initiation of protein synthesis within mammalian cells. These RNA elements (about 450 nt) contain extensive secondary structure including a hairpin loop with a conserved GNRA motif. Such loops are important in RNA-RNA and RNA-protein interactions. Plasmids that express dicistronic mRNAs of the structure GUS/IRES/HOOK have been constructed. The HOOK sequence encodes a cell-surface-targeted protein (sFv); the translation of this open reading frame within mammalian cells from these dicistronic mRNAs requires a functional IRES element. Cells that express the sFv can be selected from nonexpressing cells. A pool of up to 256 mutant encephalomyocarditis virus IRES elements was generated by converting the wild-type hairpin loop sequence (GCGA) to NNNN. Following transfection of this pool of mutants into COS-7 cells, plasmids were recovered from selected sFv-expressing cells. These DNAs were amplified in Escherichia coli and transfected again into COS-7 cells for further cycles to enrich for plasmids encoding functional IRES elements. The sequence of individual selected IRES elements was determined. All functional IRES elements had a tetraloop with a 3' terminal A residue. Optimal IRES activity, assayed in vitro and within cells, was obtained from plasmids encoding an IRES with the hairpin loop sequence fitting a RNRA consensus. In contrast, IRES elements containing YCYA tetraloops were severely defective.
Robertson
M E
ME
BBSRC Institute for Animal Health, Pirbright, Working, Surrey, United Kingdom.
Seamons
R A
RA
Belsham
G J
GJ
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002460:Cell Line; D017124:Conserved Sequence; D004686:Encephalomyocarditis virus / Q000235:genetics*; D006801:Humans; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism*; D017423:Sequence Analysis, RNA; D014162:Transfection; D014616:Vaccinia virus / Q000378:metabolism
D012333:RNA, Messenger; D012367:RNA, Viral