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Mass spectrometry and biochemical analysis of RNA polymerase II: targeting by protein phosphatase-1

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Abstract

Transcription of eukaryotic genes is regulated by phosphorylation of serine residues of heptapeptide repeats of the carboxy-terminal domain (CTD) of RNA polymerase II (RNAPII). We previously reported that protein phosphatase-1 (PP1) dephosphorylates RNAPII CTD in vitro and inhibition of nuclear PP1-blocked viral transcription. In this article, we analyzed the targeting of RNAPII by PP1 using biochemical and mass spectrometry analysis of RNAPII-associated regulatory subunits of PP1. Immunoblotting showed that PP1 co-elutes with RNAPII. Mass spectrometry approach showed the presence of U2 snRNP. Co-immunoprecipitation analysis points to NIPP1 and PNUTS as candidate regulatory subunits. Because NIPP1 was previously shown to target PP1 to U2 snRNP, we analyzed the effect of NIPP1 on RNAPII phosphorylation in cultured cells. Expression of mutant NIPP1 promoted RNAPII phosphorylation suggesting that the deregulation of cellular NIPP1/PP1 holoenzyme affects RNAPII phosphorylation and pointing to NIPP1 as a potential regulatory factor in RNAPII-mediated transcription.

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Acknowledgments

This study was supported by Department of Education and Science of Russian Federation grant No. 02.740.11.5032 from July 20th 2009. This study was also supported by NIH Research Grants 2 R25 HL003679-08 funded by the National Heart, Lung, and Blood Institute and The Office of Research on Minority Health; NIH 1SC1GM082325-01 from National Institute of General Medicine and RCMI-NIH 2G12RR003048 from the Research Centers in Minority Institutions (RCMI) Program (Division of Research Infrastructure, National Center for Research Resources, NIH). The authors thank Dr. Mathieu Bollen and Dr. Monique Beullens (Catholic University of Leuven, Belgium) for gifts of antibodies and NIPP1 expression vectors and for critical comments and suggestions. The authors would like to thank members of Dr. Victor Gordeuk’s laboratory at the Center for Sickle Cell Disease at Howard University for valuable discussions.

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Authors and Affiliations

  1. Center for Molecular Physiology, Children’s National Medical Center, Washington, DC, USA

    Marina Jerebtsova

  2. St. Petersburg Research Institute of Influenza, St. Petersburg, Russia

    Sergei A. Klotchenko, Vladimir V. Egorov, Aram A. Shaldzhyan, Maria V. Sergeeva & Andrei V. Vasin

  3. St. Petersburg State Polytechnical University, St. Petersburg, Russia

    Tatiana O. Artamonova, Evgeny A. Zatulovskiy, Olga A. Temkina, Mikhail G. Petukhov, Mikhail A. Khodorkovskii & Yuri N. Orlov

  4. Department of Medicine, Center for Sickle Cell Disease, Howard University, 1840 7th Street, N.W. HURB1, Suite 202, Washington, DC, 20001, USA

    Tatiana Ammosova, Kareem Washington & Sergei Nekhai

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  1. Marina Jerebtsova
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Correspondence to Sergei Nekhai.

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Jerebtsova, M., Klotchenko, S.A., Artamonova, T.O. et al. Mass spectrometry and biochemical analysis of RNA polymerase II: targeting by protein phosphatase-1. Mol Cell Biochem 347, 79–87 (2011). https://doi.org/10.1007/s11010-010-0614-3

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