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Molecular and Cellular Biology, June 2005, p. 4397-4405, Vol. 25, No. 11
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.11.4397-4405.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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

Keith E. Giles{dagger}, and Karen L. Beemon*

Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218

Received 16 December 2004/ Returned for modification 5 January 2005/ Accepted 7 March 2005

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.

* Corresponding author. Mailing address: Department of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218. Phone: (410) 516-7289. Fax: (410) 516-7292. E-mail: KLB{at}jhu.edu.

{dagger} Present address: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892.

Molecular and Cellular Biology, June 2005, p. 4397-4405, Vol. 25, No. 11
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.11.4397-4405.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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