Mass spectrometry and biochemical analysis of RNA polymerase II: targeting by protein phosphatase-1

Abstract

Transcription of eukaryotic genes is regulated by phosphorylation of serine residues of heptapeptide repeats of the carboxyterminal domain (CTD) of RNA polymerase II (RNAPII). We previously reported that protein phosphatase-1 (PP1) dephosphorylates RNAPII CTD in vitro and inhibition of nuclear PP1-blocked viral transcription. In this article, we analyzed the targeting of RNAPII by PP1 using biochemical and mass spectrometry analysis of RNAPII-associated regulatory subunits of PP1. Immunoblotting showed that PP1 co-elutes with RNAPII. Mass spectrometry approach showed the presence of U2 snRNP. Co-immunoprecipitation analysis points to NIPP1 and PNUTS as candidate regulatory subunits. Because NIPP1 was previously shown to target PP1 to U2 snRNP, we analyzed the effect of NIPP1 on RNAPII phosphorylation in cultured cells. Expression of mutant NIPP1 promoted RNAPII phosphorylation suggesting that the deregulation of cellular NIPP1/PP1 holoenzyme affects RNAPII phosphorylation and pointing to NIPP1 as a potential regulatory factor in RNAPII-mediated transcription.

Understanding the antagonism of retinoblastoma protein dephosphorylation by PNUTS provides insights into the PP1 regulatory code

“…Although the position of β14 varies depending on the PP1 regulatory protein bound, this is the first time, to our knowledge, that PP1 loop 21 …”

“…S11A). PNUTS binding results in a ∼3.5-Å shift of the PP1 C-terminal strand away from helix αB and the C-terminal groove and a second ∼3.5-Å shift of PP1 loop 21 …”

“…shown to associate with RNA polymerase II (RNAPII) at active sites of transcription and enhance the dephosphorylation of the RNAPII C-terminal domain (CTD) residue Ser5 (20,21). Remarkably, despite its role in these processes, the molecular function of PNUTS is still largely uncharacterized.…”

Expression of a Protein Phosphatase 1 Inhibitor, cdNIPP1, Increases CDK9 Threonine 186 Phosphorylation and Inhibits HIV-1 Transcription

“…RNA polymerase II could be a third PP1 substrate; we have previously shown that the C-terminal domain of RNA polymerase II is indeed dephosphorylated by PP1 (39). Our recent study showed that NIPP1 can serve as an RNA polymerase II-targeting subunit of PP1 (40), and thus altered RNA polymerase II dephosphorylation could also be considered as a potential inhibitory mechanism. Interestingly, the results presented here indicate that despite the expression of cdNIPP1, cells remained viable even though the association of CDK9 with 7SK RNA was increased.…”

Phosphatase 1 Nuclear Targeting Subunit Is an Essential Regulator of M-phase Entry, Maintenance, and Exit

“…As shown in Fig. 1A, Xenopus Pnuts is well conserved to the human homolog, containing the same set of functional domains, including the middle RVX(F/W)P motif that binds PP1 phosphatase (20,21,27), the YLP motif that associates with TRF2 and modulates telomere stability (25), the N-terminal RNA-binding motif that is potentially related to its function in transcriptional control (21,35,36), and the C-terminal zinc finger motif that has not been functionally characterized. To examine the role of Pnuts in M-phase exit, recombinant Pnuts proteins with GST or MBP tag were purified and added to Xenopus extracts at severalfold over endogenous Pnuts level (Figs.…”