RPRD1A and RPRD1B are human RNA polymerase II C-terminal domain scaffolds for Ser5 dephosphorylation

Abstract

SUMMARY The RNA polymerase II (RNAPII) carboxyl-terminal domain (CTD) heptapeptide repeats (Y1-S2-P3-T4-S5-P6-S7) undergo dynamic phosphorylation and dephosphorylation during the transcription cycle to recruit factors that regulate transcription, RNA processing and chromatin modification. We show here that RPRD1A and RPRD1B form homodimers and heterodimers through their coiled-coil domains and interact preferentially via CTD interaction domains (CIDs) with CTD repeats phosphorylated at S2 and S7. Our high resolution crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an interesting example of cross-talk between different CTD modifications, our data also indicate that RPRD1A and RPRD1B associate directly with RPAP2 phosphatase and, by interacting with CTD repeats where phospho-S2 and/or phospho-S7 bracket a phospho-S5 residue, serve as CTD scaffolds to coordinate the dephosphorylation of phospho-S5 by RPAP2.

Structure and dynamics of the RNAPII CTDsome with Rtt103

“…These findings indicate that the Rtt103 CID binds a longer CTD stretch than previously reported (29). Accommodation of the core P 6a S 7a Y 1b S 2b P 3b T 4b S 5b stretch of the CTD in the CID binding pocket is highly conserved among CID-CTD peptide complexes (26,27,37,38). In contrast, the conformation of the upstream A B 1.…”

“…First, we determined that Rtt103 is capable of dimerizing in free form via a coiled-coil domain. Several CID-containing proteins are known to have multimerization regions such as the Nrd1-Nab3 heterodimerization region (34), coiled-coil region in Pcf11 (35), and coiled-coil regions in RPRD1A, RPRD1B, and RPRD2 (27,36). The RPRD1A-RPRD1B heterodimer binds to multiple pS 2 -CTD repeats and exposes the pS 5 sites on the CTD, which stimulates the activity of RPAP2 pS 5 -CTD phosphatase.…”

“…and especially downstream region of the CTD peptide on the CID surface varies among CID-CTD peptide complexes (26,27,37,38). In our solution NMR structure, the pS 2 pS 7 -CTD peptide makes multiple contacts with a conserved Arg116 residue and exits the binding pocket between helices α4 and α7, thereby occupying almost the whole conserved surface of the CID.…”

“…The heterodimer composed of the human proteins RPRD1A and RPRD1B was found to bind the CTD, thereby stimulating the recruitment and phosphatase activity of RPAP2 (pS 5 -CTD-phosphatase) (27). These findings led to the proposal of a model in which the CTD and accessory molecules form a high-order arrangement dubbed the "CTDsome" (27).…”

“…A β-spiral conformation would allow the CTD chain with a length of 100 Å to fold into a compact structure, which corresponds to the observed densities in low-resolution electron microscopy images of RNAPII (20). The heterodimer composed of the human proteins RPRD1A and RPRD1B was found to bind the CTD, thereby stimulating the recruitment and phosphatase activity of RPAP2 (pS 5 -CTD-phosphatase) (27). These findings led to the proposal of a model in which the CTD and accessory molecules form a high-order arrangement dubbed the "CTDsome" (27).…”