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
Summary
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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