Function and Control of RNA Polymerase II C-Terminal Domain Phosphorylation in Vertebrate Transcription and RNA Processing

Abstract

The C-terminal domain of the RNA polymerase II largest subunit (the Rpb1 CTD) is composed of tandem heptad repeats of the consensus sequence Y¹S²P³T⁴S⁵P⁶S⁷. We reported previously that Thr 4 is phosphorylated and functions in histone mRNA 3′-end formation in chicken DT40 cells. Here, we have extended our studies on Thr 4 and to other CTD mutations by using these cells. We found that an Rpb1 derivative containing only the N-terminal half of the CTD, as well as a similar derivative containing all-consensus repeats (26r), conferred full viability, while the C-terminal half, with more-divergent repeats, did not, reflecting a strong and specific defect in snRNA 3′-end formation. Mutation in 26r of all Ser 2 (S2A) or Ser 5 (S5A) residues resulted in lethality, while Ser 7 (S7A) mutants were fully viable. While S2A and S5A cells displayed defects in transcription and RNA processing, S7A cells behaved identically to 26r cells in all respects. Finally, we found that Thr 4 was phosphorylated by cyclin-dependent kinase 9 in cells and dephosphorylated both in vitro and in vivo by the phosphatase Fcp1.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.00181-14.

ACKNOWLEDGMENTS

We thank our lab members, especially Emanuel Rosonina, for discussion and critical reading of the manuscript. We thank Stephane Larochelle and Robert Fisher for CDK9 baculoviruses and Patrick Cramer for the Fcp1 expression vector.

This work was supported by NIH grant R01GM097174.