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
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–
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. 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
)
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,
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. 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
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,
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and large numbers of splicing factors are concentrated in nuclear speckles, some of which occur at highly active transcription sites
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–
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. 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.
The nucleus contains diverse phase-separated condensates that compartmentalize and concentrate biomolecules with distinct physicochemical properties. Here, we investigated whether condensates concentrate small-molecule cancer therapeutics such that their pharmacodynamic properties are altered. We found that antineoplastic drugs become concentrated in specific protein condensates in vitro and that this occurs through physicochemical properties independent of the drug target. This behavior was also observed in tumor cells, where drug partitioning influenced drug activity. Altering the properties of the condensate was found to affect the concentration and activity of drugs. These results suggest that selective partitioning and concentration of small molecules within condensates contributes to drug pharmacodynamics and that further understanding of this phenomenon may facilitate advances in disease therapy.
Highlights d Signaling factors incorporate into Mediator condensates at super-enhancers d b-Catenin IDRs are required for both phase separation and target gene activation d Both condensate interactions and TF interactions contribute to b-catenin localization
MeCP2 (methyl CpG binding protein 2) is a key component of constitutive heterochromatin, which plays important roles in chromosome maintenance and transcriptional silencing 1-3 . Mutations in MeCP2 cause Rett syndrome (RTT) [3][4][5] , a postnatal progressive neurodevelopmental disorder associated with severe mental disability and autism-like symptoms that manifests in girls during early childhood. Heterochromatin, long considered a dense and relatively static structure 1,2 , *
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