SR PROTEIN SPLICING FACTORS INTERACT WITH THE ROUS-SARCOMA VIRUS NEGATIVE REGULATOR OF SPLICING ELEMENT

Retroviral replication requires that a portion of the primary transcri pts generated from proviral DNA be spliced to serve as mRNA for the en velope protein and in Rous sarcoma virus as src mRNA, However, a subst antial amount of full-length RNA must be maintained in an unspliced fo rm, as the unspliced RNA serves both as mRNA for structural proteins a nd 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 sequence s within the RNA of Rous sarcoma virus play a role in preserving a lar ge 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 thi s novel element blocks splicing at a distance, we set out to identify host cell factors that interact specifically with this inhibitory sequ ence, In this study, proteins from nuclear extracts with molecular mas ses 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 identified in an electrophoretic mobility shift assa y, All but p55 have biochemical properties consistent with SR protein splicing factors, and some, but not all, of the total SR proteins puri fied from HeLa cells cross-link specifically to the NRS, The strongest cross-linking SR protein is SRp30a/b, which is composed of the splici ng factors SF2/ASF and SC35, The NRS specifically binds bacterially ex pressed 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 fo r RNA splicing may be exploited by retroviruses to block splicing.