Open AccessAn RNA Splicing Enhancer-Like Sequence Is a Component of a Splicing Inhibitor Element from Rous Sarcoma Virus
Author(s) -
Lisa McNally,
Mark McNally
Publication year - 1998
Publication title -
molecular and cellular biology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.14
H-Index - 327
eISSN - 1067-8824
pISSN - 0270-7306
DOI - 10.1128/mcb.18.6.3103
Subject(s) - rna splicing , biology , enhancer , exonic splicing enhancer , sr protein , protein splicing , microbiology and biotechnology , intron , rous sarcoma virus , rna , minigene , splicing factor , alternative splicing , ribonucleoprotein , messenger rna , genetics , gene expression , gene
The accumulation in infected cells of large amounts of unspliced viral RNA for use as mRNA and genomic RNA is a hallmark of retrovirus replication. The negative regulator of splicing (NRS) is a longcis -acting RNA element in Rous sarcoma virus that contributes to unspliced RNA accumulation through splicing inhibition. One of two critical sequences located in the NRS 3′ region resembles a minor class 5′ splice site and is required for U11 small nuclear ribonucleoprotein (snRNP) binding to the NRS. The second is a purine-rich region in the 5′ half that interacts with the splicing factor SF2/ASF. In this study we investigated the possibility that this purine-rich region provides an RNA splicing enhancer function required for splicing inhibition. In vitro, the NRS acted as a potent, orientation-dependent enhancer ofDrosophila doublesex pre-mRNA splicing, and enhancer activity mapped to the purine-rich domain. Analysis of a number of site-directed and deletion mutants indicated that enhancer activity was diffusely located throughout a 60-nucleotide area but only the activity associated with a short region previously shown to bind SF2/ASF correlated with efficient splicing inhibition. The significance of the enhancer activity to splicing inhibition was demonstrated by using chimeras in which two authentic enhancers (ASLV and FP) were substituted for the native NRS purine region. In each case, splicing inhibition in transfected cells was restored to levels approaching that observed for the NRS. The observation that a nonfunctional version of the FP enhancer (FPD) that does not bind SF2/ASF also fails to block splicing when paired with the NRS 3′ region supports the notion that SF2/ASF binding to the NRS is relevant, but other SR proteins may substitute if an appropriate binding site is supplied. Our results are consistent with a role for the purine region in facilitated snRNP binding to the NRS via SF2/ASF.