| Transcription of eukaryotic protein-encoding genes is mediated by RNA polymerase II (RNAPII). The C-terminal domain (CTD) of RNAPII is required for enhancer-driven transcription, including transcription from cytomegalovirus (CMV) and human immunodeficiency virus (HIV) promoters. Phosphorylation of the CTD is required for promoter clearance and transcript elongation and also orchestrates co- or post-transcriptional events such as mRNA capping, splicing, polyadenylation, and transcriptional termination. Control of RNAPII-dependent HIV-1 transcription depends on the regulation of CTD phosphorylation. Hence, identification of CTD phosphatases may lead to novel therapies for targeting and regulating events in HIV-1 transcription. In our study we investigated whether protein phosphatase-1 (PP1) may function as a CTD phosphatase. Phosphatase assays, in vitro and in cell culture, were used to research the dephosphorylation of RNAPII by PP1. Size exclusion and affinity chromatography, as well as immunoprecipitation were used to investigate the association of PP1 with RNAPII. Transfection assays were used to deliver PP1 inhibitors into cultured cells, and gene expression analysis was used to determine genes responsive to the inhibition of PP1. We showed that PP1 dephosphorylates CTD in vitro and determined that PP1 co-purifies with RNAPII as part of the same macromolecular complex. We found that PP1 is targeted to RNAPII via NIPP1 or PP1 Nuclear Targeting Subunit (PNUTS) in cultured cells. We found that expression of inactive NIPP1 alters the dephosphorylation of RNAPII CTD and induces neuregulin-1 (NRG-1) gene expression in cultured cells. From these results, we concluded that PP1 might dephosphorylate RNAPII-CTDo in vitro and in cultured cells and that PP1 may affect transcription of cellular genes such as NRG-l. |
