SR Protein and snRNP Requirements for Assembly of the Rous Sarcoma Virus Negative Regulator of Splicing Complexin Vitro

Only a fraction of retroviral primary transcripts are spliced to subgenomic mRNAs; the unspliced transcripts are transported to the cytoplasm for packaging into virions and for translation of the gag and pol genes. We identified cis-acting sequences within the gag gene of Rous sarcoma virus (RSV) which negatively regulate splicing in vivo. Mutations were generated downstream of the splice donor (base 397) in the intron of a proviral clone of RSV. Deletion of bases 708 to 800 or 874 to 987 resulted in a large increase in the level of spliced RSV RNA relative to unspliced RSV RNA. This negative regulator of splicing (nrs) also inhibited splicing of a heterologous splice donor and acceptor pair when inserted into the intron. The nrs element did not affect the level of spliced RNA by increasing the rate of transport of the unspliced RNA to the cytoplasm but interfered more directly with splicing. To investigate the possible role of gag proteins in splicing, we studied constructs carrying frameshift mutations in the gag gene. While these mutations, which caused premature termination of gag translation, did not affect the level of spliced RSV RNA, they resulted in a large decrease in the accumulation of unspliced RNA in the cytoplasm.

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Regulation of Rous sarcoma virus RNA splicing and stability.

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4858 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4858-4867 ◽

Cited By ~ 66

Author(s):

S Arrigo ◽

K Beemon

Keyword(s):

Rous Sarcoma Virus ◽

Rna Splicing ◽

Negative Regulator ◽

Splice Donor ◽

Acceptor Pair ◽

Cis Acting ◽

Rous Sarcoma ◽

Donor And Acceptor ◽

Sarcoma Virus

Only a fraction of retroviral primary transcripts are spliced to subgenomic mRNAs; the unspliced transcripts are transported to the cytoplasm for packaging into virions and for translation of the gag and pol genes. We identified cis-acting sequences within the gag gene of Rous sarcoma virus (RSV) which negatively regulate splicing in vivo. Mutations were generated downstream of the splice donor (base 397) in the intron of a proviral clone of RSV. Deletion of bases 708 to 800 or 874 to 987 resulted in a large increase in the level of spliced RSV RNA relative to unspliced RSV RNA. This negative regulator of splicing (nrs) also inhibited splicing of a heterologous splice donor and acceptor pair when inserted into the intron. The nrs element did not affect the level of spliced RNA by increasing the rate of transport of the unspliced RNA to the cytoplasm but interfered more directly with splicing. To investigate the possible role of gag proteins in splicing, we studied constructs carrying frameshift mutations in the gag gene. While these mutations, which caused premature termination of gag translation, did not affect the level of spliced RSV RNA, they resulted in a large decrease in the accumulation of unspliced RNA in the cytoplasm.

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

Journal of Virology ◽

10.1128/jvi.00919-07 ◽

2007 ◽

Vol 81 (20) ◽

pp. 11208-11217 ◽

Cited By ~ 22

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.

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Characterization of an RNP Complex That Assembles on the Rous Sarcoma Virus Negative Regulator of Splicing Element

Nucleic Acids Research ◽

10.1093/nar/24.24.4962 ◽

1996 ◽

Vol 24 (24) ◽

pp. 4962-4968 ◽

Cited By ~ 5

Author(s):

C. R. Cook ◽

M. T. McNally

Keyword(s):

Rous Sarcoma Virus ◽

Negative Regulator ◽

Rous Sarcoma ◽

Rnp Complex ◽

Sarcoma Virus

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Position dependence of the rous sarcoma virus negative regulator of splicing element reflects proximity to a 5′ splice site

Virology ◽

10.1016/s0042-6822(03)00378-7 ◽

2003 ◽

Vol 313 (2) ◽

pp. 629-637

Author(s):

Yuedi Wang ◽

Mark T McNally

Keyword(s):

Splice Site ◽

Rous Sarcoma Virus ◽

Negative Regulator ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Position Dependence

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A cellular protein, hnRNP H, binds to the negative regulator of splicing element from Rous sarcoma virus.

Journal of Biological Chemistry ◽

10.1016/s0021-9258(18)46743-9 ◽

2001 ◽

Vol 276 (3) ◽

pp. 2310

Author(s):

Brent L. Fogel ◽

Mark T. McNally

Keyword(s):

Rous Sarcoma Virus ◽

Cellular Protein ◽

Negative Regulator ◽

Rous Sarcoma ◽

Sarcoma Virus

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

Journal of Virology ◽

10.1128/jvi.73.3.2385-2393.1999 ◽

1999 ◽

Vol 73 (3) ◽

pp. 2385-2393 ◽

Cited By ~ 32

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.

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