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

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.

ACKNOWLEDGMENTS

We thank Yingying Li for technical assistance, Tim Nilsen for helpful discussions, Massimo Caputi for aid with affinity selection, Melissa Moore for antibody against human Prp8, and Yun-Xing Wang for T7 polymerase.

This work was supported by National Institutes of Health research grant RO1 CA238796-16 to K.L.B. K.E.G. was supported in part by National Institutes of Health predoctoral training grant T32GM07231.