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

The phosphorylation of a normal cellular protein of molecular weight 34,000 (34K) is enhanced in Rous sarcoma virus-transformed chicken embryo fibroblasts apparently as a direct consequence of the phosphotransferase activity of the Rous sarcoma virus-transforming protein pp60src. We have prepared anti-34K serum by using 34K purified from normal fibroblasts to confirm that the transformation-specific phosphorylation described previously occurs on a normal cellular protein and to further characterize the nature of the protein. In this communication, we also show that the phosphorylation of 34K is also increased in cells transformed by either Fujinami or PRCII sarcoma virus, two recently characterized avian sarcoma viruses whose transforming proteins, although distinct from pp60src, are also associated with phosphotransferase activity. Moreover, comparative fingerprinting of tryptic phosphopeptides shows that the major site of phosphorylation of 34K is the same in all three cases.

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Identification of Phosphotyrosine-Containing Proteins in Untransformed and Rous Sarcoma Virus-Transformed Chicken Embryo Fibroblasts

Molecular and Cellular Biology ◽

10.1128/mcb.2.6.653 ◽

1982 ◽

Vol 2 (6) ◽

pp. 653-665 ◽

Cited By ~ 41

Author(s):

Ricardo Martinez ◽

Kenji D. Nakamura ◽

Michael J. Weber

Keyword(s):

Protein Kinases ◽

Rous Sarcoma Virus ◽

Chicken Embryo ◽

Cellular Protein ◽

Transformed Cells ◽

Phosphoamino Acid ◽

Rous Sarcoma ◽

Sarcoma Virus ◽

Embryo Fibroblasts ◽

Chicken Embryo Fibroblasts

Phosphorylation on tyrosine residues mediated by pp60srcappears to be a primary biochemical event leading to the establishment of the transformed phenotype in Rous sarcoma virus (RSV)-infected cells. To identify the cellular proteins that undergo tyrosine phosphorylation during transformation, a32P-labeled RSV-transformed chicken embryo cell extract was analyzed by electrophoresis on a polyacrylamide gel. After slicing the gel into approximately 60 slices, phosphoamino acid analyses were carried out on the protein recovered from each gel slice. Phosphotyrosine was found in every gel slice, with two major peaks of this phosphoamino acid aroundMr's of 59 and 36 kilodaltons. When the same analysis was performed with cells infected with a transformation-defectivesrcdeletion mutant of RSV (tdNY101), significant and reproducible peaks of phosphotyrosine were found in only 2 of 60 gel slices. These gel slices corresponded toMr's of 42 and 40 kilodaltons. Identical results were obtained with normal uninfected chicken embryo fibroblasts. We conclude from these observations that pp60srcor the combined action of pp60srcand pp60src-activated cellular protein kinases cause the tyrosine-specific phosphorylation of a very large number of cellular polypeptides in RSV-transformed cells. In addition, untransformed cells appear to possess one or more active tyrosine-specific protein kinases which are responsible for the phosphorylation of a limited number of proteins. These proteins are different from the major phosphotyrosine-containing proteins of the transformed cells.

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Regulation of Rous sarcoma virus RNA splicing and stability

Molecular and Cellular Biology ◽

10.1128/mcb.8.11.4858-4867.1988 ◽

1988 ◽

Vol 8 (11) ◽

pp. 4858-4867 ◽

Cited By ~ 1

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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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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Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src.

Molecular and Cellular Biology ◽

10.1128/mcb.5.5.1058 ◽

1985 ◽

Vol 5 (5) ◽

pp. 1058-1066 ◽

Cited By ~ 69

Author(s):

H Iba ◽

F R Cross ◽

E A Garber ◽

H Hanafusa

Keyword(s):

Rous Sarcoma Virus ◽

Cell Transformation ◽

Spontaneous Mutation ◽

Cellular Protein ◽

Cell Protein ◽

Wild Type ◽

Low Level ◽

Rous Sarcoma ◽

Src Gene ◽

Sarcoma Virus

We have previously found that Rous sarcoma virus variants in which the viral src (v-src) gene is replaced by the cellular src (c-src) gene have no transforming activity. In this study, we analyzed the basis for the inability of the p60c-src overproduced by these variants to transform cells. Phosphorylations of tyrosine residues in total cell protein or in cellular 34K protein are known to be markedly enhanced upon infection with wild-type Rous sarcoma virus. We found that these tyrosine phosphorylations were only slightly increased in the c-src-containing virus-infected cells, whereas both levels were significantly increased by infection with wild-type Rous sarcoma virus, or transforming mutant viruses which are derived from c-src-containing viruses by spontaneous mutation. Phosphorylation at tyrosine 416 of p60 itself was also extremely low in overproduced p60c-src and high in p60s of transforming mutant viruses. In immunoprecipitates with monoclonal antibody, the overproduced p60c-src had much lower casein tyrosine kinase activity than did p60v-src. We previously showed that p60 myristylation and plasma membrane localization may be required for cell transformation. p60c-src was similar to transforming p60s in these properties. These results strongly suggest that the low level of tyrosine phosphorylation by overproduced p60c-src accounts for its inability to transform cells.

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