CaltechAUTHORS
Published August 22, 2019 | Version Supplemental Material + Accepted Version
Journal Article Open

Pol II phosphorylation regulates a switch between transcriptional and splicing condensates

Creators

  • 1. ROR icon Whitehead Institute for Biomedical Research
  • 2. ROR icon Massachusetts Institute of Technology
  • 3. ROR icon University of Illinois at Chicago
  • 4. ROR icon St. Jude Children's Research Hospital
  • 5. ROR icon University of Colorado Boulder

Abstract

The synthesis of pre-mRNA by RNA polymerase II (Pol II) involves the formation of a transcription initiation complex, and a transition to an elongation complex. The large subunit of Pol II contains an intrinsically disordered C-terminal domain that is phosphorylated by cyclin-dependent kinases during the transition from initiation to elongation, thus influencing the interaction of the C-terminal domain with different components of the initiation or the RNA-splicing apparatus. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain. Both the transcription-initiation machinery and the splicing machinery can form phase-separated condensates that contain large numbers of component molecules: hundreds of molecules of Pol II and mediator are concentrated in condensates at super-enhancers, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites. Here we investigate whether the phosphorylation of the Pol II C-terminal domain regulates the incorporation of Pol II into phase-separated condensates that are associated with transcription initiation and splicing. We find that the hypophosphorylated C-terminal domain of Pol II is incorporated into mediator condensates and that phosphorylation by regulatory cyclin-dependent kinases reduces this incorporation. We also find that the hyperphosphorylated C-terminal domain is preferentially incorporated into condensates that are formed by splicing factors. These results suggest that phosphorylation of the Pol II C-terminal domain drives an exchange from condensates that are involved in transcription initiation to those that are involved in RNA processing, and implicates phosphorylation as a mechanism that regulates condensate preference.

Additional Information

© 2019 Nature Publishing Group. Received 17 October 2018. Accepted 10 July 2019. Published 07 August 2019. We thank I. A. Klein, J. Schuijers, C. H. Li, E. L. Coffey and other members in the Young laboratory for helpful discussions, W. Salmon of the W. M. Keck Microscopy Facility, T. Volkert, J. Love and S. Gupta of the Whitehead Genomics Core facility for technical assistance, M. Stubna for help with droplet and biochemical assays and S. L. McKnight, I. Kwon and M. Kato for CTD constructs. The work was supported by NIH grant GM123511 (R.A.Y.), NSF grant PHY1743900 (R.A.Y. and P.A.S.), NIH GM117370 and GM110064 (D.J.T.), NIH grant R01-GM034277 (P.A.S.), Cancer Research Institute Irvington Fellowship (Y.E.G.), Damon Runyon Cancer Research Foundation Fellowship (2309-17) (B.R.S.), Hope Funds for Cancer Research fellowship (B.J.A.), Swedish Research Council Postdoctoral Fellowship (VR 2017-00372) (A.B.), the German Research Foundation DFG Postdoctoral Fellowship SP 1680/1-1 (J.-H.S.), DE 3069/1-1 (T.-M.D.), NIH T32 GM008759 (C.B.F.) and funds from Novo Nordisk (R.A.Y. and P.A.S.). These authors contributed equally: Yang Eric Guo, John C. Manteiga. Author Contributions. Y.E.G. and R.A.Y. conceived the project. Y.E.G., J.C.M. and R.A.Y. organized the studies and wrote the manuscript. Y.E.G. and B.R.S. performed in vitro droplet formation assays. J.C.M., A.D. and A.B. performed immunofluorescence experiments. J.E.H. and K.S. developed and performed computational analyses. B.R.S., Y.E.G, B.J.A. and J.C.M. performed ChIP and analysed data. N.M.H. purified recombinant proteins. Y.E.G., J.E.H. and L.K.A. generated cell lines. J.-H.S. and A.V.Z. performed lattice light-sheet microscopy and analysis. T.-M.D., J.K.R., C.B.F. and D.J.T. purified human mediator. Y.E.G., J.C.M. and J.E.H. generated constructs. J.E.H. performed live cell imaging. J.E.H. and T.I.L. contributed to writing the manuscript. P.A.S. and I.I.C. provided input into experimental design and interpretation. R.A.Y. supervised the project with the help from T.I.L. All authors contributed to editing the manuscript. Data availability. Datasets generated in this study have been deposited in the Gene Expression Omnibus under accession number GSE120656. Uncropped gel images can be found in Supplementary Fig. 1. Code availability. All custom code used in this study is available upon request. Competing interests. R.A.Y. is a founder and shareholder of Syros Pharmaceuticals, Camp4 Therapeutics, Omega Therapeutics and Dewpoint Therapeutics. P.A.S. is a member of the board and shareholder in Syros and a member of the Scientific Advisory Board of Dewpoint. B.J.A. and T.I.L. are shareholders of Syros Pharmaceuticals. T.I.L. is a consultant to Camp4 Therapeutics and I.I.C. is a consultant to Dewpoint Therapeutics. All other authors declare no competing interests.

Attached Files

Accepted Version - nihms-1534331.pdf

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Supplemental Material - 41586_2019_1464_MOESM2_ESM.pdf

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Additional details

Identifiers

PMCID
PMC6706314
Eprint ID
106648
DOI
10.1038/s41586-019-1464-0
Resolver ID
CaltechAUTHORS:20201112-143947946

Related works

Describes
10.1038/s41586-019-1464-0 (DOI)
http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE120656 (URL)

Funding

NIH
GM123511
NSF
PHY-1743900
NIH
GM117370
NIH
GM110064
NIH
R01-GM034277
Cancer Research Institute
Damon Runyon Cancer Research Foundation
2309-17
Hope Funds for Cancer Research
Swedish Research Council
VR 2017-00372
Deutsche Forschungsgemeinschaft (DFG)
SP 1680/1-1
Deutsche Forschungsgemeinschaft (DFG)
DE 3069/1-1
NIH Predoctoral Fellowship
T32 GM008759
Novo Nordisk

Dates

Created
2020-11-16
Created from EPrint's datestamp field
Updated
2021-11-16
Created from EPrint's last_modified field