Retroviral splicing suppressor sequesters a 3' splice site in a 50S aberrant splicing complex.
Retroviral replication requires both spliced and unspliced mRNAs. Splicing suppression of avian retroviral RNA depends in part upon a cis-acting element within the gag gene called the negative regulator of splicing (NRS). The NRS, linked to a downstream intron and exon (NRS-Ad3'), was not capable of splicing in vitro. However, a double-point mutation in the NRS pseudo-5' splice site sequence converted it into a functional 5' splice site. The wild-type (WT) NRS-Ad3' transcript assembled an approximately 50S spliceosome-like complex in vitro; its sedimentation rate was similar to that of a functional spliceosome formed on the mutant NRS-Ad3' RNA. The five major spliceosomal snRNPs were observed in both complexes by affinity selection. In addition, U11 snRNP was present only in the WT NRS-Ad3' complex. Addition of heparin to these complexes destabilized the WT NRS-Ad3' complex; it was incapable of forming a B complex on a native gel. Furthermore, the U5 snRNP protein, hPrp8, did not cross-link to the NRS pseudo-5' splice site, suggesting that the tri-snRNP complex was not properly associated with it. We propose that this aberrant, stalled spliceosome, containing U1, U2, and U11 snRNPs and a loosely associated tri-snRNP, sequesters the 3' splice site and prevents its interaction with the authentic 5' splice site upstream of the NRS.
Giles
Keith E
KE
Department of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218, USA.
Beemon
Karen L
KL
D002352:Carrier Proteins / Q000378:metabolism; D016153:Genes, Suppressor; D015750:Genes, gag / Q000235:genetics*; D006801:Humans; D017354:Point Mutation; D022821:RNA Splice Sites / Q000235:genetics / Q000502:physiology*; D012326:RNA Splicing / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism; D016601:RNA-Binding Proteins; D012190:Retroviridae / Q000235:genetics*; D017411:Ribonucleoproteins, Small Nuclear / Q000378:metabolism*; D017381:Spliceosomes / Q000378:metabolism*; D013489:Suppression, Genetic; D014158:Transcription, Genetic; D014779:Virus Replication / Q000235:genetics
D002352:Carrier Proteins; C115374:PRPF8 protein, human; D022821:RNA Splice Sites; D012333:RNA, Messenger; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D017411:Ribonucleoproteins, Small Nuclear