A small yeast RNA selectively inhibits internal initiation of translation programmed by poliovirus RNA: specific interaction with cellular proteins that bind to the viral 5'-untranslated region.
We have purified, sequenced, and prepared a synthetic clone of a small (60-nucleotide) RNA molecule from the yeast Saccharomyces cerevisiae that had previously been isolated on the basis of its ability to selectively block the translation of poliovirus mRNA. RNA derived from the clone by transcription with T7 RNA polymerase appears to block translation initiation by internal ribosome entry (cap independent) but does not significantly affect cap-dependent translation. Deletion analysis of the poliovirus 5'-untranslated region (5'-UTR) has shown that yeast inhibitor RNA (I-RNA) requires internal ribosome entry site sequences to inhibit the translation of poliovirus RNA in vitro. Using a bicistronic RNA construct, we show that I-RNA preferentially inhibits translation by internal ribosome entry. Gel retardation and UV cross-linking studies demonstrate that I-RNA specifically binds proteins which interact with RNA secondary structures within the poliovirus 5'-UTR presumably involved in internal initiation. Specifically, purified I-RNA competes with virus RNA structures within the 5'-UTR which bind a cellular protein with an approximate molecular mass of 52 kDa. Finally, when transfected into HeLa cells, I-RNA efficiently inhibits the replication of poliovirus RNA presumably by inhibiting translation of the input virus RNA.
Das
S
S
Department of Microbiology and Immunology, UCLA School of Medicine 90024-1747.
Coward
P
P
Dasgupta
A
A
D001483:Base Sequence; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017955:Poliovirus / Q000235:genetics*; D011485:Protein Binding; D014176:Protein Biosynthesis; D012331:RNA, Fungal / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D012367:RNA, Viral / Q000235:genetics* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics*
D012331:RNA, Fungal; D012367:RNA, Viral; D016601:RNA-Binding Proteins