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Splicing and transcription-associated proteins PSF and p54nrb/NonO bind to the RNA polymerase II CTD

Published online by Cambridge University Press:  06 September 2002

ANDREW EMILI ,
MICHAEL SHALES ,
SUSAN McCRACKEN ,
WEIJUN XIE ,
PHILIP W. TUCKER ,
RYUJI KOBAYASHI ,
BENJAMIN J. BLENCOWE  and
C. JAMES INGLES
ANDREW EMILI
Affiliation:
Banting and Best Department of Medical Research, C.H. Best Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
MICHAEL SHALES
Affiliation:
Banting and Best Department of Medical Research, C.H. Best Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada
SUSAN McCRACKEN
Affiliation:
Banting and Best Department of Medical Research, C.H. Best Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada
WEIJUN XIE
Affiliation:
Department of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78705, USA
PHILIP W. TUCKER
Affiliation:
Department of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78705, USA
RYUJI KOBAYASHI
Affiliation:
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA Present address: Department of Molecular Pathology, University of Texas, MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas 77030, USA.
BENJAMIN J. BLENCOWE
Affiliation:
Banting and Best Department of Medical Research, C.H. Best Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
C. JAMES INGLES
Affiliation:
Banting and Best Department of Medical Research, C.H. Best Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
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Abstract

The carboxyl-terminal domain (CTD) of the largest subunit of eukaryotic RNA polymerase II (pol II) plays an important role in promoting steps of pre-mRNA processing. To identify proteins in human cells that bind to the CTD and that could mediate its functions in pre-mRNA processing, we used the mouse CTD expressed in bacterial cells in affinity chromatography experiments. Two proteins present in HeLa cell extract, the splicing and transcription-associated factors, PSF and p54nrb/NonO, bound specifically and could be purified to virtual homogeneity by chromatography on immobilized CTD matrices. Both hypo- and hyperphosphorylated CTD matrices bound these proteins with similar selectivity. PSF and p54nrb/NonO also copurified with a holoenzyme form of pol II containing hypophosphorylated CTD and could be coimmunoprecipitated with antibodies specific for this and the hyperphosphorylated form of pol II. That PSF and p54nrb/NonO promoted the binding of RNA to immobilized CTD matrices suggested these proteins can interact with the CTD and RNA simultaneously. PSF and p54nrb/NonO may therefore provide a direct physical link between the pol II CTD and pre-mRNA processing components, at both the initiation and elongation phases of transcription.

Keywords

affinity chromatographypre-mRNA processingprotein complexestranscription
Type
REPORTS
Information
RNA , Volume 8 , Issue 9 , September 2002 , pp. 1102 - 1111
Copyright
2002 RNA Society

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