title: SR protein and snRNP requirements for assembly of the Rous sarcoma virus negative regulator of splicing complex in vitro.
abstract: Retroviruses use unspliced RNA as mRNA for expression of virion structural proteins and as genomic RNA; the full-length RNA often constitutes the majority of the viral RNA in an infected cell. Maintenance of this large pool of unspliced RNA is crucial since even a modest increase in splicing efficiency can lead to impaired replication. In Rous sarcoma virus, the negative regulator of splicing (NRS) was identified as a cis element that negatively impacts splicing of viral RNA. Components of the splicing apparatus appear to be involved in splicing inhibition since binding of a number of splicing factors (snRNPs and SR proteins) and assembly of a large complex (NRS-C) in nuclear extracts correlate with NRS-mediated splicing inhibition. In determining the requirements for NRS complex assembly, we show that NRS-C assembly can be reconstituted by addition of total SR proteins to an S100 extract that lacks these factors. Of the purified SR proteins tested, SF2/ASF was functional in NRS-C assembly, whereas SC35 and SRp40 were not. The participation of snRNPs in NRS-C assembly was addressed by selectively depleting individual snRNPs with oligonucleotides and RNase H or by sequestering critical snRNA domains with 2'-O-methyl RNA oligonucleotides. The results indicate that in addition to U11 snRNP, U1 snRNP and SR proteins, but not U2 snRNP, are involved in NRS-C assembly.
authors.affiliation: Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226, USA.
authors.forename: C R
authors.initials: CR
authors.lastname: Cook
authors.forename: M T
authors.initials: MT
authors.lastname: McNally
mesh_terms: D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics / Q000502:physiology*; D002467:Cell Nucleus / Q000378:metabolism; D005809:Genes, Regulator; D008836:Micrococcal Nuclease; D012326:RNA Splicing; D012333:RNA, Messenger / Q000096:biosynthesis*; D012367:RNA, Viral / Q000096:biosynthesis*; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D014158:Transcription, Genetic; D014771:Virion / Q000235:genetics / Q000502:physiology; D014779:Virus Replication
chemical_list: D012333:RNA, Messenger; D012367:RNA, Viral; D017411:Ribonucleoproteins, Small Nuclear; D008836:Micrococcal Nuclease