The role of overlapping U1 and U11 5' splice site sequences in a negative regulator of splicing.
Splicing of Rous sarcoma virus RNA is regulated in part by a cis-acting intronic RNA element called the negative regulator of splicing (NRS). An NRS mutant affecting nt 916-923 disrupts U11 snRNP binding and reduces NRS activity (Gontarek et al., 1993, Genes & Dev 7:1926-1936). However, we observed that a U15' splice site-like sequence, which overlapped the U11 site, was also disrupted by this mutation. To determine whether the U1 or the U11 site was essential for NRS activity, we analyzed twelve additional mutants involving nt 915-926. All mutations that disrupted the potential base pairing between U1 snRNA and the NRS reduced NRS activity, including single point mutations at nt 915, 916, and 919. The point mutation at nt 919 was partially suppressed by a compensatory base change mutation in U1 snRNA. In contrast, a mutation which strengthened the potential base pairing between the U1 site and the NRS increased NRS activity. Surprisingly, mutations that specifically targeted the U115' splice site consensus sequence increased the levels of unspliced RNA, suggesting U11 binding plays an antagonistic role to NRS activity. We propose that U1 snRNP binding to the NRS inhibits splicing and is regulated by U11 snRNP binding to the overlapping sequence. Competition between U1 and U11 snRNPs would result in the appropriate balance of spliced to unspliced RNAs for optimal viral replication. Further, a virus mutated in the U1/U11 region of the NRS was found to have delayed replication.
Hibbert
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CS
Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
Gontarek
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RR
Beemon
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KL
D001358:Avian Sarcoma Viruses / Q000235:genetics; D020029:Base Pairing / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D015967:Gene Expression Regulation, Viral / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D009154:Mutation / Q000235:genetics; D012326:RNA Splicing / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D016601:RNA-Binding Proteins / Q000235:genetics; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000235:genetics*; D017411:Ribonucleoproteins, Small Nuclear / Q000235:genetics; D014162:Transfection; D014779:Virus Replication
D012367:RNA, Viral; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; D017411:Ribonucleoproteins, Small Nuclear; C098762:U11 small nuclear ribonucleoprotein, human