RNA



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REPORTS

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


ANDREW  EMILI  a1 a2, MICHAEL  SHALES  a1, SUSAN  McCRACKEN  a1, WEIJUN  XIE  a3, PHILIP W.  TUCKER  a3, RYUJI  KOBAYASHI  a4 p1, BENJAMIN J.  BLENCOWE  a1 a2 and C. JAMES  INGLES  a1 a2 c1
a1 Banting and Best Department of Medical Research, C.H. Best Institute, University of Toronto, Toronto, Ontario M5G 1L6, Canada
a2 Department of Molecular and Medical Genetics, University of Toronto, Toronto, Ontario M5S 1A8, Canada
a3 Department of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78705, USA
a4 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA

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.

(Received March 6 2002)
(Revised March 18 2002)
(Accepted June 14 2002)


Key Words: affinity chromatography; pre-mRNA processing; protein complexes; transcription.

Correspondence:
c1 Reprint requests to: C. James Ingles, Banting and Best Department of Medical Research, C.H. Best Institute, University of Toronto, 112 College Street, Toronto, Ontario M5G 1L6, Canada; e-mail: cj.ingles@utoronto.ca
p1 Present address: Department of Molecular Pathology, University of Texas, MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas 77030, USA.