Abstract
The RNA polymerase II (RNAPII) C-terminal domain (CTD) heptapeptide repeats (1-YSPTSPS-7) 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 RNAPII CTD repeats phosphorylated at S2 and S7. Crystal structures of the RPRD1A, RPRD1B and RPRD2 CIDs, alone and in complex with RNAPII CTD phosphoisoforms, elucidate the molecular basis of CTD recognition. In an 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.
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Acknowledgements
We thank D.Y. Zhao, X. Wu and K. Liu for technical assistance, J. Moffat (University of Toronto) for providing the shRNAs and D. Eick (Helmholtz-Zentrum, Munich) for providing antibodies. This work was supported by grants from the Canadian Institutes of Health Research (CIHR MOP-258357) (to J.F.G. and A.E.), the Ontario Research Fund (to J.F.G. and A.E.), the US National Institutes of Health (GM099714) (to A.L.M.), the Wellcome Trust (092483/Z/10/Z), Edward Penley Abraham (to S.M.) and the Canada Foundation for Innovation (CFI) (to J.F.G. and A.E.), and by a CIHR Postdoctoral Fellowship (to Z.N.), Oxford Stem Cell Institute Studentship (to O.V.K.) and Ontario Graduate Scholarship (to J.B.O.). The Structural Genomics Consortium is a registered charity (number 1097737) that receives funds from AbbVie, Boehringer Ingelheim, CFI, CIHR, Genome Canada through the Ontario Genomics Institute (OGI-055), GlaxoSmithKline, Janssen, Lilly Canada, the Novartis Research Foundation, the Ontario Ministry of Economic Development and Innovation, Pfizer, Takeda and the Wellcome Trust (092809/Z/10/Z) (to C.H.A. and J.M.). Results in this report are partially derived from work performed at Argonne National Laboratory, Structural Biology Center at the Advanced Photon Source. Argonne is operated by University of Chicago, Argonne, for the US Department of Energy, Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.
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Z.N. and J.F.G. conceived the project and designed the experiments. Z.N. and C.X. performed protein expression, purification and crystallization experiments and analyzed the structure data. W.T. and M.E.B. conducted crystallographic data collection, structure determination and refinement. Z.N., X.G., G.O.H., O.V.K., E.M., G.Z., H.G., W.-H.W.K., J.L., P.Y., J.B.O., C.W., P.L., G.A.S., H. He and H. Huang conducted experiments. S.S.S., A.E., S.M., A.L.M., C.H.A. and J.M. guided the experiments. All authors commented on the manuscript. Z.N. analyzed the data. Z.N. and J.F.G. wrote the manuscript. J.M. and J.F.G. supervised the project.
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Integrated supplementary information
Supplementary Figure 2 Dimerization of RPRDs. Related to Figure 4.
(a) Size exclusion chromatography analysis of the indicated recombinant proteins purified from E. coli. (b) Dynamic light scattering measurement of the molecular weights of the indicated recombinant proteins purified from E. coli. The ratios of measured vs monomer molecular weight are shown at the top of each bar. Error bars: s.d. from three technical replicates. (c) SDS-polyacrylamide gel analysis showing the indicated recombinant proteins cross-linked with the indicated concentration of suberic acid-bis-(3-sulfo-N-hydroxysuccinimide ester) (BS3). Putative multimers are labeled.
Supplementary Figure 3 RPRD1A interacts with RPRD1B via coiled-coil domain in vitro. Related to Figure 4.
Glutathione-S-transferase (GST) pull-down showing the interactions between GST-tagged RPRD1A and His-tagged RPRD1B, its CID or its coiled-coil domain expressed in E. coli.
Supplementary Figure 4 RPAP2 phosphatase activity in vitro. Related to Figure 6.
(a) In vitro phosphatase assays using 6,8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) substrate showing the effects of RPRD1A and RPRD1B on RPAP2 (1-334) activity. The reaction includes 1 μM RPAP2 (1-334), 10 μM DiFMUP and indicated concentration of RPRD1A or RPRD1B. The fluorescence level induced by RPAP2 (1-334) was set as 100%. Error bars: s.d. from four technical replicates. (b) In vitro DiFMUP phosphatase assays showing the effects on RPAP2 phosphatase activity by the phosphatase inhibitor vanadate. RPAP2 (1-334): 2 μM. DiFMUP: 10 μM. Vanadate: 1 mM. The fluorescence level induced by RPAP2 (1-334) was set as 100%. *: p<0.01 as compared with the no inhibitor controls (two-tailed Student’s t-test). Error bars: s.d. from five technical replicates. (c) In vitro DiFMUP phosphatase assays showing the effects on RPAP2 phosphatase activity by indicated metals. RPAP2 (1-334): 2 μM. DiFMUP: 10 μM. Indicated metals: 10 mM. The fluorescence level induced by RPAP2 (1-334) in the absence of added metals was set as 100%. *: p<0.01 as compared with the no metal controls (one-way ANOVA Tukey's Multiple Comparison test). Error bars: s.d. from six technical replicates. (d) In vitro phosphatase ELISAs showing the effects of vanadate and the indicated metals on RPRD1A-stimulated RPAP2 phosphatase activity on the indicated biotinylated CTD peptide. The reactions contain three µM RPAP2, 10 μM RPRD1A and biotinylated S2-5-2P CTD peptide, with or without 1 mM vanadate or 10 mM of the indicated metals, incubated at 37 °C for 3 hr, followed by measurement of the phosphorylation levels using the S5P monoclonal antibody (3E8). No protein controls were set as 100%. *: p<0.01 as compared with the no inhibitor controls (two-tailed Student’s t-test). Error bars; s.d. from four technical replicates. (e) In vitro phosphatase ELISAs showing the effects on the antibody recognition at S5P by adjacent S7P. The reactions contain the indicated biotinylated CTD peptides incubated with 3 µM RPAP2 and 10 µM RPRD1A at 37 °C for 3 hours. Phosphorylation levels were measured using the indicated antibodies. *: p<0.01 (two-tailed Student’s t-test). Error bars: s.d. from three technical replicates. (f) In vitro phosphatase ELISAs assays showing the effects of RPRD1A or RPRD1B stimulating RPAP2 phosphatase activity on S5P located between two S7P-containing CTD repeats. The reactions contain the indicated biotinylated CTD peptides, indicated proteins incubated at 37 °C for 3 hours, followed by measurement of the phosphorylation levels using the indicated antibodies. No protein controls were set as 100%. *: p<0.01 (two-tailed Student’s t-test) as compared to no protein control. Error bars: s.d. from four technical replicates.
Supplementary Figure 5 Regulation of the phosphorylation level of CTD S5 in vivo. Related to Figure 6.
(a) Chromatin immunoprecipitation (ChIP) experiments using the indicated antibodies showing the effects on S5P levels at various promoters by shRNA-mediated knock-down of RPAP2 in HEK293 cells. *: p < 0.05 compared with shGFP (two-tailed Student’s t-test). Error bars: s.d. from three biological replicates. (b) Western blotting using the indicated antibodies showing the effects on the global S5P and S2P levels by shRNA-mediated knock-down of RPAP2 in HEK293 cells. WT: no lentiviral shRNA infected cells. (c) ChIP experiments using the indicated antibodies showing the effects on S5P levels at various promoters or near promoter regions by siRNA-mediated knocking down RPRD1A in HeLa cell extracts. ACTB (β-ACTIN ): +300 bp region. The S5P levels were normalized to those of RNAPII (detected by N20 antibody as in Fig. 5e). *: p < 0.05 compared with no siRNA transfection control (two-tailed Student’s t-test). Error bars: s.d. from three biological replicates. The bar for β -ACTIN indicates the range of two biological replicates.
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Supplementary Text and Figures
Supplementary Figures 1–5 and Supplementary Tables 1–4 (PDF 1227 kb)
Supplementary Data Set
Original gel images for western blots (PDF 1030 kb)
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Ni, Z., Xu, C., Guo, X. et al. RPRD1A and RPRD1B are human RNA polymerase II C-terminal domain scaffolds for Ser5 dephosphorylation. Nat Struct Mol Biol 21, 686–695 (2014). https://doi.org/10.1038/nsmb.2853
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DOI: https://doi.org/10.1038/nsmb.2853
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