Document Detail


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.
MedLine Citation:
PMID:  10496218     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
M E Robertson; R A Seamons; G J Belsham
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Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  RNA (New York, N.Y.)     Volume:  5     ISSN:  1355-8382     ISO Abbreviation:  RNA     Publication Date:  1999 Sep 
Date Detail:
Created Date:  1999-10-05     Completed Date:  1999-10-05     Revised Date:  2009-11-18    
Medline Journal Info:
Nlm Unique ID:  9509184     Medline TA:  RNA     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  1167-79     Citation Subset:  IM    
Affiliation:
BBSRC Institute for Animal Health, Pirbright, Working, Surrey, United Kingdom.
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MeSH Terms
Descriptor/Qualifier:
Animals
Base Sequence
COS Cells
Cell Line
Conserved Sequence
Encephalomyocarditis virus / genetics*
Humans
Models, Genetic
Molecular Sequence Data
Mutagenesis
Nucleic Acid Conformation*
Plasmids / genetics
Polymerase Chain Reaction
RNA, Messenger / analysis,  genetics
RNA, Viral / genetics*,  metabolism*
Ribosomes / genetics*,  metabolism*
Sequence Analysis, RNA
Transfection
Vaccinia virus / metabolism
Chemical
Reg. No./Substance:
0/RNA, Messenger; 0/RNA, Viral
Comments/Corrections

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