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MBC in Press, published online ahead of print February 8, 2006
Mol. Biol. Cell 10.1091/mbc.E05-08-0726

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Submitted on August 5, 2005
Revised on January 26, 2006
Accepted on January 30, 2006

Splicing Speckles Are Not Reservoirs of RNA Polymerase II, but Contain an Inactive Form, Phosphorylated on Serine2 Residues of the CTD

Sheila Q. Xie,* Sonya Martin,*{dagger} Pascale V. Guillot,*{ddagger} David L. Bentley,{sect} and Ana Pombo*

*MRC Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, United Kingdom; {sect}Department of Biochemistry and Molecular Genetics, UCHSC, University of Colorado School of Medicine, Aurora, CO 80045

Monitoring Editor: A. Gregory Matera

"Splicing speckles" are major nuclear domains rich in components of the splicing machinery and polyA+ RNA. Although speckles contain little detectable transcriptional activity, they are found preferentially associated with specific mRNA-coding genes and gene-rich R bands, and accumulate some unspliced pre-mRNAs. RNA polymerase II transcribes mRNAs and is required for splicing, with some reports suggesting that the inactive complexes are stored in splicing speckles. Using ultrathin cryosections to improve optical resolution and preserve nuclear structure, we find that all forms of polymerase II are present, but not enriched, within speckles. Inhibition of polymerase activity shows that speckles do not act as major storage sites for inactive polymerase II complexes, but contain a stable pool of polymerase II phosphorylated on Serine2 residues of the CTD, which is transcriptionally inactive and may have roles in spliceosome assembly or post-transcriptional splicing of pre-mRNAs. Paraspeckle domains lie adjacent to speckles, but little is known about their protein content or putative roles in the expression of the speckle-associated genes. We find that paraspeckles are transcriptionally inactive but contain polymerase II, which remains stably associated upon transcriptional inhibition, when paraspeckles reorganize around nucleoli in the form of caps.


Present addresses: {dagger}Tenovus Research Laboratory, University of Southampton, Southampton General Hospital, Tremona Road, Southampton SO15 5PA, United Kingdom; {ddagger}Institute of Reproductive and Developmental Biology, Imperial College London, Du Cane Road, London W12 0NN, United Kingdom.

Address correspondence to: Ana Pombo (ana.pombo{at}csc.mrc.ac.uk)




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