Interaction between the negative regulator of splicing element and a 3 ' splice site: Requirement for U1 small nuclear ribonucleoprotein and the 3 ' splice site branch point/pyrimidine tract

The negative regulator of splicing (NRS) from Rous sarcoma virus suppresses viral RNA splicing and is one of several cis elements that account for the accumulation of large amounts of unspliced RNA for use as gag-pol mRNA and progeny virion genomic RNA. The NRS can also inhibit splicing of heterolog ous introns in vivo and in vitro. Previous data showed that the splicing fa ctors SF2/ASF and U1, U2, and U11 small nuclear ribonucleoproteins (snRNPs) bind the NRS, and a correlation was established between SF2/ASF and U11 bi nding and activity, suggesting that these factors are important for functio n. These observations, and the finding that a large spliceosome-like comple x (NRS-C) assembles on NRS RNA in nuclear extract, led to the proposal that the NRS is recognized as a minor-class 5' splice site. One model to explai n NRS splicing inhibition holds that the NRS interacts nonproductively with and sequesters U2-dependent 3' splice sites, In this study, we provide evi dence that the NRS interacts with an adenovirus 3' splice site. The interac tion was dependent on the integrity of the branch point and pyrimidine trac t of the 3' splice site, and it was sensitive to a mutation that was previo usly shown to abolish U11 snRNP binding and NRS function, However, further mutational analyses of NRS sequences have identified a U1 binding site that overlaps the U11 site, and the interaction with the 3' splice site correla ted with U1, not U11, binding. These results show that the NRS can interact with a 3' splice site and suggest that U1 is of primary importance for NRS splicing inhibition.