Retroviral Splicing Suppressor Sequesters a 3′ Splice Site in a 50S Aberrant Splicing Complex

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Abstract
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 ∼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.