Abstract
Abstract: :
Purpose: Pnn and its associated SR–family members, co–localize and interact in nuclear speckles in the context of corneal epithelial cells, as we previously demonstrated. Interestingly, Pnn is also capable of binding to co–repressor CtBP, a multifunctional transcriptional regulator implicated in processes governing tumorigenesis and embryonic development. Transcriptional co–repressors bridge basal transcription machinery to enzymatic complexes capable of maintaining the silenced chromatin state. Our earlier observations imply that Pnn can exhibit a pronounced nuclear redistribution in response to CtBP overexpression. Here, we investigate the effect of CtBP overexpression on the nuclear distribution of Pnn and SR proteins in corneal epithelial and HeLa cells as an initial attempt to characterize the function of CtBP in context of mRNA processing events. Methods: Cultured corneal epithelial cells HCE–T were transfected with epitope–tagged CtBP and/or Pnn expression vectors and the localization of endogenous and exogenous Pnn and SR–proteins was assessed using specific antibodies. Likewise, HeLa cells engineered to stably express exogenous CtBP–HA–Flag were utilized to analyze nuclear distribution of Pnn and SR proteins. Results: Transfection of HCE–T cells using CtBP–Flag expression vector followed by immunostaining for Pnn and SR proteins demonstrated that in response to CtBP overexpression Pnn and SR proteins redistributed from the nuclear speckles and acquired a more diffuse nucleoplasmic localization pattern. Similar nucleoplasmic distributions of Pnn and SR–proteins were observed in a HeLa cell line engineered to overexpress exogenous CtBP. Furthermore, HCE–Ts overexpressing exogenous CtBP in the cytoplasm exhibited Pnn and SR proteins accumulated in cytoplasm. Conclusions:Our data suggests that co–repressor CtBP can affect not only nuclear distribution of Pnn but also SR protein family members. In light of recent reports demonstrating the interaction of co–repressors and splicing proteins, it is possible that CtBP might participate in regulation of splicing through influencing nuclear localization of pre–mRNA processing factors such as Pnn and SR family members. (Supported by NIH grant EY07883)
Keywords: cornea: basic science • gene/expression • transcription