Abstract

SummaryThe carboxy-terminal domain (CTD) of the large subunit of RNA polymerase II (Pol II) comprises multiple heptapeptide repeats of the consensus Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Reversible phosphorylation of Ser2, Ser5, and Ser7 during the transcription cycle mediates the sequential recruitment of transcription/RNA processing factors. Phosphorylation of Ser7 is required for recruitment of the gene type-specific Integrator complex to the Pol II-transcribed small nuclear (sn)RNA genes. Here, we show that RNA Pol II-associated protein 2 (RPAP2) specifically recognizes the phospho-Ser7 mark on the Pol II CTD and also interacts with Integrator subunits. siRNA-mediated knockdown of RPAP2 and mutation of Ser7 to alanine cause similar defects in snRNA gene expression. In addition, we show that RPAP2 is a CTD Ser5 phosphatase. Taken together, our results indicate that during transcription of snRNA genes, Ser7 phosphorylation facilitates recruitment of RPAP2, which in turn both recruits Integrator and dephosphorylates Ser5.

Highlights

  • In human cells, transcription of protein-coding genes and most small nuclearRNA genes is carried out by RNA polymerase (Pol) II

  • The presence of the IIa and IIo forms, corresponding to hypo- and hyperphosphorylated carboxy-terminal domain (CTD), respectively, suggests that RNA Pol II-associated protein 2 (RPAP2) associates with transcriptionally active polymerase II (Pol II)

  • This analysis revealed that RPAP2 and Rpb1 elute together in a large complex (LC) peaking in fractions 11–13, while a significant fraction of F-RPAP2 elutes as a smaller complex (SC), peaking at fractions 4–6, devoid of Rpb1 but containing Int4, Int5, Int6, and Int7

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Summary

Introduction

Transcription of protein-coding genes and most small nuclear (sn)RNA genes is carried out by RNA polymerase (Pol) II. It is well established that the CTD acts as a molecular platform allowing the transcription/RNA processing factors to be recruited to the transcribing polymerase at the right point of the transcription cycle (Buratowski, 2003; Egloff and Murphy, 2008; Perales and Bentley, 2009). Dynamic dephosphorylation of Ser and Ser is thought to make a significant contribution to the changes in CTD phosphorylation patterns during the transcription cycle and is essential for recycling Pol II (Buratowski, 2009; Egloff and Murphy, 2008). The evolutionarily conserved protein Fcp, which is essential in yeast, dephosphorylates phospho-Ser preferentially (Hausmann and Shuman, 2002), whereas Ssu and the more recently described Rtr in yeast, and SCP1 in mammals, dephosphorylate Ser (Krishnamurthy et al, 2004; Mosley et al, 2009; Yeo et al, 2003)

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