Splicing mediates the activity of four putative cellular internal ribosome entry sites.
A growing number of cellular mRNAs are thought to possess internal ribosome entry sites (IRESs), sequences that permit translation of a transcript independent of its 5' end and cap structure. Although dicistronic assays are the canonical method of testing sequences for IRES activity, they may produce false-positive results if unanticipated monocistronic RNAs arise from the dicistronic construct used. Using a dicistronic reporter system and a green fluorescent protein-tagged retrovirus to evaluate six previously reported cellular IRESs, we found that four contain 3' splice sites whose activity was required for apparent IRES function and which resulted in formation of monocistronic transcripts by splicing. Bioinformatic analysis revealed that the 3' splice sites identified in three of these putative IRESs are used in their native mRNAs and that the fourth is likely an artifactual sequence created during cDNA cloning. Our findings demonstrate a need for reexamination of other reported cellular IRESs by using careful RNA structural analysis to rule out splicing as the source of perceived IRES activity.
Baranick
Brian T
BT
Interdepartmental Program in Molecular Biology, Department of Molecular and Medical Pharmacology, University of California, 675 Charles E. Young Drive South, MRL 1551, Los Angeles, CA 90095-7019, USA.
Lemp
Nathan A
NA
Nagashima
Jill
J
Hiraoka
Kei
K
Kasahara
Noriyuki
N
Logg
Christopher R
CR
D000818:Animals; D016477:Artifacts; D001483:Base Sequence; D003001:Cloning, Molecular; D039603:Eukaryotic Initiation Factor-4G / Q000235:genetics / Q000378:metabolism; D005796:Genes / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D010957:Plasmids; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012367:RNA, Viral / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D012270:Ribosomes / Q000235:genetics*; D019076:Transgenes; D051636:X-Linked Inhibitor of Apoptosis Protein / Q000235:genetics / Q000378:metabolism
D039603:Eukaryotic Initiation Factor-4G; C099506:RBM3 protein, human; D022821:RNA Splice Sites; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D051636:X-Linked Inhibitor of Apoptosis Protein; D049452:Green Fluorescent Proteins