JCB logo
amgmicro.com
  Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents
This Article
Right arrow Full Text
Right arrow Full Text (PDF, 505K)
Right arrow PPT slides of all figures
Right arrow Alert me when this article is cited
Right arrow Citation Map
Services
Right arrow Email this article
Right arrow Similar articles in this journal
Right arrow Alert me to new content in the JCB
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via CrossRef
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Kim, E.
Right arrow Articles by Warren, S. L.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Kim, E.
Right arrow Articles by Warren, S. L.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Facebook   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

© The Rockefeller University Press, 0021-9525/1997//19 $5.00
The Journal of Cell Biology, Volume 136, Number 1, , 1997 19-28

Article

Splicing Factors Associate with Hyperphosphorylated RNA Polymerase II in the Absence of Pre-mRNA



Euikyung Kim, Lei Du*, David B. Bregman, and Stephen L. Warren

Department of Pathology and * Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06510

The carboxy-terminal domain (CTD) of the largest subunit of RNA polymerase II (Pol II) contains multiple tandem copies of the consensus heptapeptide, TyrSerProThrSerProSer. Concomitant with transcription initiation the CTD is phosphorylated. Elongating polymerase has a hyperphosphorylated CTD, but the role of this modification is poorly understood. A recent study revealed that some hyperphosphorylated polymerase molecules (Pol IIo) are nonchromosomal, and hence transcriptionally unengaged (Bregman, D.B., L. Du, S. van der Zee, S.L. Warren. 1995. J. Cell Biol. 129: 287–298). Pol IIo was concentrated in discrete splicing factor domains, suggesting a possible relationship between CTD phosphorylation and splicing factors, but no evidence beyond immunolocalization data was provided to support this idea. Here, we show that Pol IIo co-immunoprecipitates with members of two classes of splicing factors, the Sm snRNPs and non-snRNP SerArg (SR) family proteins. Significantly, Pol IIo's association with splicing factors is maintained in the absence of pre-mRNA, and the polymerase need not be transcriptionally engaged. We also provide definitive evidence that hyperphosphorylation of Pol II's CTD is poorly correlated with its transcriptional activity. Using monoclonal antibodies (mAbs) H5 and H14, which are shown here to recognize phosphoepitopes on Pol II's CTD, we have quantitated the level of Pol IIo at different stages of the cell cycle. The level of Pol IIo is similar in interphase and mitotic cells, which are transcriptionally active and inactive, respectively. Finally, complexes containing Pol IIo and splicing factors can be prepared from mitotic as well as interphase cells. The experiments reported here establish that hyperphosphorylation of the CTD is a good indicator of polymerase's association with snRNP and SR splicing factors, but not of its transcriptional activity. Most importantly, the present study suggests that splicing factors may associate with the polymerase via the hyperphosphorylated CTD.

Abbreviations used in this paper: CTD, carboxy-terminal domain of RNA polymerase II; MIG, mitotic interchromatin granule cluster; NuMA, nuclear mitotic apparatus protein; Pol IIa, unphosphorylated largest RNA polymerase II subunit; Pol II LS, largest subunit of RNA polymerase II; Pol IIo, hyperphosphorylated largest RNA polymerase II subunit; Sm sn RNP, Smith antigen-containing small nuclear ribonucleoprotein; SR, serine-arginine dipeptide repeat motif.

The work was supported by the Council for Tobacco Research (#3881) and National Institutes of Health (NIH) (K08-CA01339) to S.L. Warren and by the NIH (F32 CA09281) to D.B. Bregman.

Please address all correspondence to S.L Warren, Brady Memorial Laboratories, Room B117, Department of Pathology, Yale University School of Medicine, P.O. Box 208023, New Haven, CT 06520-8023. Tel.: (203) 737-2247. Fax: (203) 785-7303. E-mail: stephen.warren{at}yale.edu


Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Facebook Facebook   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?


This article has been cited by other articles:



  Home | Help | Feedback | Subscriptions | Archive | Search | Table of Contents