Pol II phosphorylation regulates a switch between transcriptional and splicing condensates

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 1 – 4 . The large subunit of Pol II contains an intrinsically disordered C-terminal domain (CTD), which is phosphorylated by cyclin-dependent kinases (CDKs) during the initiation-to-elongation transition, thus influencing the CTD’s interaction with different components of the initiation or the RNA splicing apparatus ( Fig. 1a ) 5 , 6 . Recent observations suggest that this model provides only a partial picture of the effects of CTD phosphorylation. Both the transcription initiation machinery and the splicing machinery can form phase-separated condensates containing large numbers of component molecules; hundreds of Pol II and Mediator molecules are concentrated in condensates at super-enhancers 7 , 8 and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites 9 – 12 . Here we investigate whether phosphorylation of the CTD regulates its incorporation into phase-separated condensates associated with transcription initiation and splicing. We find that the hypophosphorylated Pol II CTD is incorporated into Mediator condensates and that phosphorylation by regulatory CDKs reduces this incorporation. We also find that the hyperphosphorylated CTD is preferentially incorporated into condensates formed by splicing factors. These results suggest that Pol II CTD phosphorylation drives an exchange from condensates involved in transcription initiation to those involved in RNA processing and implicates phosphorylation as a mechanism to regulate condensate preference.

Preferred conformations in the disordered region of human CPEB3, a functional amyloid linked to memory consolidation

“…1A) (Alston et al, 2004). The far UV CD spectrum of the hCPEB3 IDR also shows the hallmarks of a disordered protein, namely a minimum near 200 nm (Denning et al, 2003). No spectral features indicative of a-helix and b-sheet; namely, minima at 208, 218 or 222 nm and no maximum at 195 nm, are evident (Sup .…”

PHF3 regulates neuronal gene expression through the new Pol II CTD reader domain SPOC

“…Coupling between elongation and RNA processing through Pol II CTD becomes even more relevant considering recent findings that the CTD undergoes liquid-liquid phase separation (LLPS) Lu et al, 2018). LLPS of unphosphorylated CTD may facilitate Pol II clustering and transcriptional bursting during transcription initiation, whereas the subsequent LLPS wave of pS2-phosphorylated CTD may trigger Pol II clustering with RNA processing factors (Guo et al, 2019). PHF3 may promote the second wave of CTD LLPS through direct binding to pS2 CTD and RNA processing factors.…”

Suppression of liquid-liquid phase separation by 1,6-hexanediol partially compromises the 3D genome organization in living cells

“…Second, we examined whether the LLPS-dependent membrane-free intranuclear compartments, such as Cajal bodies and splicing speckles, were affected by 1,6-HD treatment. These compartments were choosen because they were reported to be sensitive to 1,6-HD (Lin et al 2016;Guo et al 2019). The indirect immunofluorescence analysis of coilin, a major proteinaceous component of Cajal bodies, showed that 1,6-HD treatment (5%, 15 min), but not the treatment with Tween 20 alone, disrupted these compartments in living cells ( Fig.…”