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Vol. 17, Issue 4, 1723-1733, April 2006

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

Sheila Q. Xie ^*, Sonya Martin ^* , Pascale V. Guillot ^* , David L. Bentley , and Ana Pombo ^*

^* Medical Research Council Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, United Kingdom; Department of Biochemistry and Molecular Genetics, University of Colorado Health Sciences Center, University of Colorado School of Medicine, Aurora, CO 80045

Submitted August 5, 2005; Revised January 26, 2006; Accepted January 30, 2006
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 they 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 that they contain a stable pool of polymerase II phosphorylated on serine² residues of the C-terminal domain, which is transcriptionally inactive and may have roles in spliceosome assembly or posttranscriptional 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.

Abbreviations used: AP, alkaline phosphatase; Br-RNA, bromo-UTP–labeled RNA; CTD, C-terminal domain; DRB, 5,6-dichloro-1--D-ribofuranosylbenzimidazole; EGFP, enhanced green fluorescent protein; IF, immunofluorescence; pol II, RNA polymerase II_A; pol II, hypophosphorylated polymerase II; pol II_O, hyperphosphorylated polymerase II; PSP, paraspeckle protein; Ser²P, phosphorylated serine² residues of the CTD; Ser⁵P, phosphorylated serine⁵ residues of the CTD; SR, serine/arginine; WB, Western blot.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Present address: Tenovus Research Laboratory, University of Southampton, Southampton General Hospital, Southampton SO15 5PA, United Kingdom

Present address: Institute of Reproductive and Developmental Biology, Imperial College London, London W12 0NN, United Kingdom.

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

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