SR protein splicing factors interact with the Rous sarcoma virus negative regulator of splicing element.
Retroviral replication requires that a portion of the primary transcripts generated from proviral DNA be spliced to serve as mRNA for the envelope protein and in Rous sarcoma virus as src mRNA. However, a substantial amount of full-length RNA must be maintained in an unspliced form, as the unspliced RNA serves both as mRNA for structural proteins and virion-associated enzymatic proteins and as genomic RNA for progeny virions. The extent of viral RNA splicing must be finely controlled, since only a narrow range in the ratio of unspliced RNA to spliced RNA is tolerated for optimal replication. A number of cis-acting sequences within the RNA of Rous sarcoma virus play a role in preserving a large pool of unspliced RNA. One such sequence, the negative regulator of splicing (NRS), is of interest because it blocks splicing but is not located near any of the splice junctions. To better understand how this novel element blocks splicing at a distance, we set out to identify host cell factors that interact specifically with this inhibitory sequence. In this study, proteins from nuclear extracts with molecular masses of 26, 36, 44, and 55 kDa were shown by UV cross-linking assays to bind the NRS preferentially. One of them, p55, was also detected in a specific complex with SR protein electrophoretic mobility shift assay. All but p55 have biochemical properties consistent with SR protein splicing factors, and some, but not all, of the total SR proteins purified from HeLa cells cross-link specifically to the NRS. The strongest cross-linking SR protein is SRp30a/b, which is composed of the splicing factors SF2/ASF and SC35. The NRS specifically binds bacterially expressed SF2/ASF, whereas nonfunctional mutants do not. Data indicating that the 36-kDa protein which cross-links in nuclear extracts is SF2/ASF are presented. The data indicate that factors normally required for RNA splicing may be exploited by retroviruses to block splicing.
McNally
L M
LM
Department of Microbiology, Medical College of Wisconsin, Milwaukee 53226, USA.
McNally
M T
MT
D001358:Avian Sarcoma Viruses / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003432:Cross-Linking Reagents; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D011687:Purines / Q000378:metabolism; D012326:RNA Splicing; D012367:RNA, Viral / Q000378:metabolism*; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; D014466:Ultraviolet Rays
D003432:Cross-Linking Reagents; D009687:Nuclear Proteins; D010750:Phosphoproteins; D011687:Purines; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D000068103:Serine-Arginine Splicing Factors; C030985:purine