Publications
Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. YE Guo, JC Manteiga, JE Henninger, BR Sabari, A Dall’Agnese, NM Hannett, JH Spille, LK Afeyan, AV Zamudio, K Shrinivas, BJ Abraham, A Boija, TM Decker, JK Rimel, CB Fant, TI Lee, II Cisse, PA Sharp, DJ Taatjes, RA Young . Nature 572, 543-548 (2019). Link
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 complex1,2,3,4. 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 apparatus5,6. Recent observations suggest that this model provides only a partial picture of the effects of phosphorylation of the C-terminal domain7,8,9,10,11,12. 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-enhancers7,8, and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites9,10,11,12. 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.
Our study on Mediator and Pol II condensates left us wondering about some of the drug treatment results. Notably, treatment with the CDK9 kinase inhibitor DRB - used then as a transcription inhibitor - led to an apparent overall decrease in the number of Pol II condensates. But some of them seemed to grow much brighter and bigger. Our collaborators at the Whitehead Institute investigated this more thoroughly and showed that Pol II CTD phosphorylation (by e.g. CDK9, but also CDK7) reduces partitioning of Pol II into Mediator droplets. Instead, the phosphorylated Pol II CTD more strongly partitions into splicing factor droplets.
Using multicolor lattice light sheet imaging we were able to confirm this finding in live cells. The emerging picture is that small molecular modification (like phosphorylation of certain residues in intrinsically disordered regions of proteins) can act as a switch to drive factors into specific condensates.
This work was covered in the following article:
