A Cellular Protein, hnRNP H, Binds to the Negative Regulator of Splicing Element from Rous Sarcoma Virus

• Published in: The Journal of biological chemistry 2000-10, Vol.275 (41), p.32371-32378
• Main Authors: Fogel, Brent L., McNally, Mark T.
• Format: Article
• Language: English
• Subjects: Avian Sarcoma Viruses - genetics; Base Sequence; Binding Sites; Gene Expression Regulation, Viral; HeLa Cells; Heterogeneous-Nuclear Ribonucleoprotein Group F-H; Heterogeneous-Nuclear Ribonucleoproteins; hnRNP H protein; Humans; Mass Spectrometry; Molecular Sequence Data; Nuclear Proteins - chemistry; Nuclear Proteins - immunology; Nuclear Proteins - isolation & purification; Nuclear Proteins - metabolism; p55 protein; Precipitin Tests; Protein Binding; Recombinant Proteins; Regulatory Sequences, Nucleic Acid - genetics; Ribonucleoproteins - chemistry; Ribonucleoproteins - genetics; Ribonucleoproteins - immunology; Ribonucleoproteins - metabolism; RNA Splicing - genetics; RNA, Viral - genetics; RNA, Viral - metabolism; RNA-Binding Proteins - chemistry; RNA-Binding Proteins - genetics; RNA-Binding Proteins - immunology; RNA-Binding Proteins - metabolism; Rous sarcoma virus; snRNP U1 protein; snRNP U11 protein; Ultraviolet Rays
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M005000200

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 acis-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 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.