SR PROTEIN AND SNRNP REQUIREMENTS FOR ASSEMBLY OF THE ROUS-SARCOMA VIRUS NEGATIVE REGULATOR OF SPLICING COMPLEX IN-VITRO

Retroviruses use unspliced RNA as mRNA for expression of virion struct ural proteins and as genomic RNA; the full-length RNA often constitute s the majority of the viral RNA in an infected cell. Maintenance of th is large pool of unspliced RNA is crucial since even a modest increase in splicing efficiency can lead to impaired replication. In Rous sarc oma virus, the negative regulator of splicing (NRS) was identified as a cis element that negatively impacts splicing of viral RNA. Component s of the splicing apparatus appear to be involved in splicing inhibiti on since binding of a number of splicing factors (snRNPs and SR protei ns) and assembly of a large complex (NRS-C) in nuclear extracts correl ate with NRS-mediated splicing inhibition. In determining the requirem ents for NRS complex assembly, we show that NRS-C assembly can be reco nstituted by addition of total SR proteins to an S100 extract that lac ks these factors. Of the purified SR proteins tested, SF2/ASF was func tional in NRS-C assembly, whereas SC35 and SRp40 were not. The partici pation of snRNPs in NRS-C assembly was addressed by selectively deplet ing individual snRNPs with oligonucleotides and RNase H or by sequeste ring critical snRNA domains with 2'-O-methyl RNA oligonucleotides. The results indicate that in addition to U11 snRNP, U1 snRNP and SR prote ins, but not U2 snRNP, are involved in NRS-C assembly. (C) 1998 Academ ic Press.