A cellular protein, hnRNP H, binds to the negative regulator of splicing element from Rous sarcoma virus.
Incomplete RNA splicing is a key feature of the retroviral life cycle. This is in contrast to the processing of most cellular pre-mRNAs, which are usually spliced to completion. In Rous sarcoma virus, splicing control is achieved in part through a cis-acting RNA element termed the negative regulator of splicing (NRS). The NRS is functionally divided into two parts termed NRS5' and NRS3', which bind a number of splicing factors. The U1 and U11 small nuclear ribonucleoproteins interact with sequences in NRS3', whereas NRS5' binds several proteins including members of the SR [corrected] family of proteins. Among the proteins that specifically bind NRS5' is a previously unidentified 55-kDa protein (p55). In this report we describe the isolation and identification of p55. The p55 binding site was localized by UV cross-linking to a 31-nucleotide segment, and a protein that binds specifically to it was isolated by RNA affinity selection and identified by mass spectrometry as hnRNP H. Antibodies against hnRNP H immunoprecipitated cross-linked p55 and induced a supershift of a p55-containing complex formed in HeLa nuclear extract. Furthermore, UV cross-linking and electrophoretic mobility shift assays indicated that recombinant hnRNP H specifically interacts with the p55 binding site, confirming that hnRNP H is p55. The possible roles of hnRNP H in NRS function are discussed.
Department of Microbiology and Molecular Genetics, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
B L
BL
Fogel
M T
MT
McNally
D001358:Avian Sarcoma Viruses / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D015967:Gene Expression Regulation, Viral; D006367:HeLa Cells; D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D011233:Precipitin Tests; D011485:Protein Binding; D012326:RNA Splicing / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D012261:Ribonucleoproteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D014466:Ultraviolet Rays
D034541:Heterogeneous-Nuclear Ribonucleoprotein Group F-H; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009687:Nuclear Proteins; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D012261:Ribonucleoproteins