Splicing and transcription-associated proteins PSF and p54nrb/NonO bind to the RNA polymerase II CTD
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.
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: firstname.lastname@example.org
p1 Present address: Department of Molecular Pathology, University of Texas, MD Anderson Cancer Center, 1515 Holcombe Blvd., Houston, Texas 77030, USA.