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S.P. Sugrue, Y. Shi, M.R. Jackson, R. Alpatov, M.E. Hunt; Isolation of the nuclear Pnn–complex from epithelial cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):1042.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: We have shown that pinin localizes to nuclear speckles and binds to several SR proteins including SRp75, SRm300 and SRrp130, which are thought to play a role in pre–mRNA processing. Pinin can also bind to the transcriptional co–repressor CtBP and potentially regulate the transcription of genes, including E–cadherin. We sought to determine the biochemical characterization the intranuclear Pnn–complex. Methods: We generated stable lines of corneal epithelial cells (HCE–T, RCB1384, K Sasaki) and HeLa cells carrying epitope–tagged Pnn via infecting cells with a recombinant retrovirus expressing a bicistronic messenger RNA containing open reading frames of Flag–HA tagged human Pnn and interleukin–2 receptor (IL–2R)–α. Infected HCE–T and HeLa cells were sorted by anti–IL–2R monoclonal antibody conjugated to magnetic beads, and the resultant Pnn–Flag–HA stable cell lines were propagated. The expression levels of the tagged–Pnn were comparable to that of endogenous Pnn. The Pnn complex was purified from nuclear extracts by tandem affinity, using anti–Flag M2 mAb– and anti–HA mAb 16b12 –conjugated agarose beads. Results: Creation of stable cell lines expressing epitope–tagged Pnn enabled the isolation and identification of Pnn–associated proteins. Western blots of the isolated complexes were performed utilizing a monoclonal antibody directed against a shared epitope found on a subset of approximately 20 non–snRNP splicing proteins. The Western blot revealed that the affinity–purified Pnn complex did contain an array of endogenous SR proteins. Prominent SR–immunoreactive bands, with Mr(K) approximately 55, 75, 100, and 200 kD, were co–purified with Pnn. These results are consistent with Pnn’s localization at the nuclear speckle. Further proteomic analyses of proteins comprising these complexes are underway. Conclusions:Taken together, Pnn is in a complex with multiple SR–rich proteins, supporting the data implicating Pnn functioning in splicing, It is tempting to speculate that Pnn may play a structural and/or functional role in the mRNA processing. It is therefore conceivable that Pnn is associated with central regulatory protein complexes that function in transcriptional regulation and pre–mRNA maturation, or spatial organization of these processes in the nucleus of epithelial cells. (Supported by NIH grant EY07883)
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