scholarly journals The Negative Regulator of Splicing Element of Rous Sarcoma Virus Promotes Polyadenylation

2006 ◽  
Vol 80 (19) ◽  
pp. 9634-9640 ◽  
Author(s):  
Jeremy E. Wilusz ◽  
Karen L. Beemon
Keyword(s):  
Splice Site ◽  
Rous Sarcoma Virus ◽  
Sr Proteins ◽  
Negative Regulator ◽  
Rous Sarcoma ◽  
Splice Site Sequence ◽  
Sarcoma Virus ◽  
Flanking Sequences ◽  
A Site

ABSTRACT The Rous sarcoma virus gag gene contains a cis-acting negative regulator of splicing (NRS) element that is implicated in viral polyadenylation regulation. To study the mechanism of polyadenylation promotion at the viral poly(A) site located over 8 kb downstream, we performed in vitro polyadenylation analysis. RNA containing only the poly(A) site and flanking sequences in the 3′ long terminal repeat (LTR) was not polyadenylated detectably in vitro; however, if the transcript contained the NRS upstream of the LTR, polyadenylation was observed. Insertion of the viral env 3′ splice site sequence between the NRS and the LTR did not alter the level of polyadenylation appreciably. We conclude that the NRS promotes polyadenylation in vitro and can do so without formation of a splicing complex with a 3′ splice site. We then explored the roles of several cellular factors in NRS-mediated polyadenylation. Mutation of the binding sites of U1 and U11 snRNPs to the NRS did not affect polyadenylation, whereas hnRNP H strongly inhibited polyadenylation. We propose a model in which hnRNP H and SR proteins compete for binding to the NRS. Bound SR proteins may bridge between the NRS and the 3′ LTR and aid in the recruitment of the 3′-end processing machinery.

scholarly journals Serine/Arginine-Rich Proteins Contribute to Negative Regulator of Splicing Element-Stimulated Polyadenylation in Rous Sarcoma Virus

2007 ◽  
Vol 81 (20) ◽  
pp. 11208-11217 ◽  
Author(s):  
Nicole L. Maciolek ◽  
Mark T. McNally
Keyword(s):  
Splice Site ◽  
Binding Sites ◽  
Rous Sarcoma Virus ◽  
Sr Proteins ◽  
Negative Regulator ◽  
Control Element ◽  
Long Distance ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
A Site

ABSTRACT Rous sarcoma virus (RSV) requires large amounts of unspliced RNA for replication. Splicing and polyadenylation are coupled in the cells they infect, which raises the question of how viral RNA is efficiently polyadenylated in the absence of splicing. Optimal RSV polyadenylation requires a far-upstream splicing control element, the negative regulator of splicing (NRS), that binds SR proteins and U1/U11 snRNPs and functions as a pseudo-5′ splice site that interacts with and sequesters 3′ splice sites. We investigated a link between NRS-mediated splicing inhibition and efficient polyadenylation. In vitro, the NRS alone activated a model RSV polyadenylation substrate, and while the effect did not require the snRNP-binding sites or a downstream 3′ splice site, SR proteins were sufficient to stimulate polyadenylation. Consistent with this, SELEX-binding sites for the SR proteins ASF/SF2, 9G8, and SRp20 were able to stimulate polyadenylation when placed upstream of the RSV poly(A) site. In vivo, however, the SELEX sites improved polyadenylation in proviral clones only when the NRS-3′ splice site complex could form. Deletions that positioned the SR protein-binding sites closer to the poly(A) site eliminated the requirement for the NRS-3′ splice site interaction. This indicates a novel role for SR proteins in promoting RSV polyadenylation in the context of the NRS-3′ splice site complex, which is thought to bridge the long distance between the NRS and poly(A) site. The results further suggest a more general role for SR proteins in polyadenylation of cellular mRNAs.

scholarly journals U1 Small Nuclear Ribonucleoprotein and Splicing Inhibition by the Rous Sarcoma Virus Negative Regulator of Splicing Element

1999 ◽  
Vol 73 (3) ◽  
pp. 2385-2393 ◽  
Author(s):  
Lisa M. McNally ◽  
Mark T. McNally
Keyword(s):  
Rous Sarcoma Virus ◽  
Sr Proteins ◽  
Negative Regulator ◽  
Rous Sarcoma ◽  
Small Nuclear Ribonucleoprotein ◽  
Virus Rna ◽  
U1 Snrnp ◽  
Sarcoma Virus ◽  
A Minor ◽  
Nuclear Ribonucleoprotein

ABSTRACT Retroviruses require both spliced and unspliced RNA for replication. Accumulation of unspliced Rous sarcoma virus RNA is facilitated in part by a negative cis element in thegag region, termed the negative regulator of splicing (NRS), which serves to repress splicing of viral RNA but can also block splicing of heterologous introns. The NRS binds components of the splicing machinery including SR proteins, U1 and U2, small nuclear ribonucleoproteins (snRNPs) of the major splicing pathway, and U11 snRNP of the minor pathway, yet splicing does not normally occur from the NRS. A mutation that abolishes U11 binding (RG11) also abrogates NRS splicing inhibition, indicating that U11 is functionally important for NRS activity and suggesting that the NRS is recognized as a minor-class 5′ splice site (5′ ss). We show here, using specific NRS mutations to disrupt U11 binding and coexpression of U11 snRNA genes harboring compensatory mutations, that the NRS U11 site is functional when paired with a minor-class 3′ ss from the human P120 gene. Surprisingly, the expectation that the same NRS mutants would be defective for splicing inhibition proved false; splicing inhibition was as good as, if not better than, that for the wild-type NRS. Comparison of these new mutations with RG11 indicated that the latter may disrupt binding of a factor(s) other than U11. Our data suggest that this factor is U1 snRNP and that a U1 binding site that overlaps the U11 site is also disrupted by RG11. Analysis of mutations which selectively disrupted U1 or U11 binding indicated that splicing inhibition by the NRS correlates most strongly with U1 snRNP. Additionally, we show that U1 binding is facilitated by SR proteins that bind to the 5′ half of the NRS, confirming an earlier proposal that this region is involved in recruiting snRNPs to the NRS. These data indicate a functional role for U1 in NRS-mediated splicing inhibition.

scholarly journals Retroviral Splicing Suppressor Requires Three Nonconsensus Uridines in a 5′ Splice Site-Like Sequence

2001 ◽  
Vol 75 (16) ◽  
pp. 7763-7768 ◽  
Author(s):  
Robert E. Paca ◽  
Catherine S. Hibbert ◽  
Christopher T. O'Sullivan ◽  
Karen L. Beemon
Keyword(s):  
Splice Site ◽  
Rous Sarcoma Virus ◽  
Negative Regulator ◽  
Rous Sarcoma ◽  
Virus Rna ◽  
U1 Snrna ◽  
Sarcoma Virus ◽  
Site Consensus

ABSTRACT Rous sarcoma virus RNA contains a negative regulator of splicing (NRS) element that aids in maintenance of unspliced RNA. The NRS binds U1 snRNA at a sequence that deviates from the 5′ splice site consensus by substitution of U's for A's at three positions: −2, +3, and +4. All three of these U's are important for NRS-mediated splicing suppression. Substitution of a single nonconsensus C or G at any of these sites diminished NRS activity, whereas substitution of a single A generated a preferred 5′ splice site within the NRS.

scholarly journals An RNA Splicing Enhancer-Like Sequence Is a Component of a Splicing Inhibitor Element from Rous Sarcoma Virus

1998 ◽  
Vol 18 (6) ◽  
pp. 3103-3111 ◽  
Author(s):  
Lisa M. McNally ◽  
Mark T. McNally
Keyword(s):  
Rous Sarcoma Virus ◽  
Rna Splicing ◽  
Negative Regulator ◽  
Rich Region ◽  
Enhancer Activity ◽  
Rous Sarcoma ◽  
Small Nuclear Ribonucleoprotein ◽  
Sarcoma Virus ◽  
Splicing Enhancer

ABSTRACT 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 of Drosophila doublesexpre-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.

Functional domains of the pp60v-src protein as revealed by analysis of temperature-sensitive Rous sarcoma virus mutants

1984 ◽  
Vol 4 (8) ◽  
pp. 1508-1514
Author(s):  
A W Stoker ◽  
P J Enrietto ◽  
J A Wyke
Keyword(s):  
Rous Sarcoma Virus ◽  
Kinase Activity ◽  
Temperature Sensitive ◽  
Tyrosine Kinase Activity ◽  
Rous Sarcoma ◽  
Heat Labile ◽  
Src Gene ◽  
Sarcoma Virus

Four temperature-sensitive (ts) Rous sarcoma virus src gene mutants with lesions in different parts of the gene represent three classes of alteration in pp60src. These classes are composed of mutants with (i) heat-labile protein kinase activities both in vitro and in vivo (tsLA27 and tsLA29), (ii) heat-labile kinases in vivo but not in vitro (tsLA33), and (iii) neither in vivo nor in vitro heat-labile kinases (tsLA32). The latter class indicates the existence of structural or functional pp60src domains that are required for transformation but do not grossly affect tyrosine kinase activity.

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