Dephosphorylation of RNA polymerase II by CTD-phosphatase FCP1 is inhibited by phospho-CTD associating proteins.

Reversible phosphorylation of the repetitive C-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit plays a key role in the progression of RNAP through the transcription cycle. The level of CTD phosphorylation is determined by multiple CTD kinases and a CTD phosphatase, FCP1. The phosphorylated CTD binds to a variety of proteins including the cis/trans peptidyl-prolyl isomerase (PPIase) Pin1 and enzymes involved in processing of the primary transcript such as the capping enzyme Hce1 and CA150, a nuclear factor implicated in transcription elongation. Results presented here establish that the dephosphorylation of hyperphosphorylated RNAP II (RNAP IIO) by FCP1 is impaired in the presence of Pin1 or Hce1, whereas CA150 has no influence on FCP1 activity. The inhibition of dephosphorylation is observed with free RNAP IIO generated by different CTD kinases as well as with RNAP IIO engaged in an elongation complex. These findings support the idea that specific phospho-CTD associating proteins can differentially modulate the dephosphorylation of RNAP IIO by steric hindrance and may play an important role in the regulation of gene expression.

TFIIF-associating carboxyl-terminal domain phosphatase dephosphorylates phosphoserines 2 and 5 of RNA polymerase II.

The carboxyl-terminal domain (CTD) of the largest RNA polymerase (RNAP) II subunit undergoes reversible phosphorylation throughout the transcription cycle. The unphosphorylated form of RNAP II is referred to as IIA, whereas the hyperphosphorylated form is known as IIO. Phosphorylation occurs predominantly at serine 2 and serine 5 within the CTD heptapeptide repeat and has functional implications for RNAP II with respect to initiation, elongation, and transcription-coupled RNA processing. In an effort to determine the role of the major CTD phosphatase (FCP1) in regulating events in transcription that appear to be influenced by serine 2 and serine 5 phosphorylation, the specificity of FCP1 was examined. FCP1 is capable of dephosphorylating heterogeneous RNAP IIO populations of HeLa nuclear extracts. The extent of dephosphorylation at specific positions was assessed by immunoreactivity with monoclonal antibodies specific for phosphoserine 2 or phosphoserine 5. As an alternative method to assess FCP1 specificity, RNAP IIO isozymes were prepared in vitro by the phosphorylation of purified calf thymus RNAP IIA with specific CTD kinases and used as substrates for FCP1. FCP1 dephosphorylates serine 2 and serine 5 with comparable efficiency. Accordingly, the specificity of FCP1 is sufficiently broad to dephosphorylate RNAP IIO at any point in the transcription cycle irrespective of the site of serine phosphorylation within the consensus repeat.

FCP1 phosphorylation by casein kinase 2 enhances binding to TFIIF and RNA polymerase II carboxyl-terminal domain phosphatase activity.

Dephosphorylation of RNA polymerase II carboxyl-terminal domain (CTD) is required to resume sequential transcription cycles. FCP1 (TFIIF-dependent CTD phosphatase 1) is the only known phosphatase targeting RNAP II CTD. Here we show that in Xenopus laevis cells, xFCP1 is a phosphoprotein. On the basis of biochemical fractionation and drug sensitivity, casein kinase 2 (CK2) is shown to be the major kinase involved in xFCP1 phosphorylation in X. laevis egg extracts. CK2 phosphorylates xFCP1 mainly at a cluster of serines centered on Ser(457). CK2-dependent phosphorylation enhances 4-fold the CTD phosphatase activity of FCP1 and its binding to the RAP74 subunit of general transcription factor TFIIF. These findings unravel a new mechanism regulating CTD phosphorylation and hence class II gene transcription.