Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II

Abstract

Hyperphosphorylation of the C-terminal domain (CTD) of the RPB1 subunit of human RNA polymerase (Pol) II is essential for transcriptional elongation and mRNA processing. The CTD contains 52 heptapeptide repeats of the consensus sequence YSPTSPS. The highly repetitive nature and abundant possible phosphorylation sites of the CTD exert special constraints on the kinases that catalyse its hyperphosphorylation. Positive transcription elongation factor b (P-TEFb)-which consists of CDK9 and cyclin T1-is known to hyperphosphorylate the CTD and negative elongation factors to stimulate Pol II elongation. The sequence determinant on P-TEFb that facilitates this action is currently unknown. Here we identify a histidine-rich domain in cyclin T1 that promotes the hyperphosphorylation of the CTD and stimulation of transcription by CDK9. The histidine-rich domain markedly enhances the binding of P-TEFb to the CTD and functional engagement with target genes in cells. In addition to cyclin T1, at least one other kinase-DYRK1A -also uses a histidine-rich domain to target and hyperphosphorylate the CTD. As a low-complexity domain, the histidine-rich domain also promotes the formation of phase-separated liquid droplets in vitro, and the localization of P-TEFb to nuclear speckles that display dynamic liquid properties and are sensitive to the disruption of weak hydrophobic interactions. The CTD-which in isolation does not phase separate, despite being a low-complexity domain-is trapped within the cyclin T1 droplets, and this process is enhanced upon pre-phosphorylation by CDK7 of transcription initiation factor TFIIH. By using multivalent interactions to create a phase-separated functional compartment, the histidine-rich domain in kinases targets the CTD into this environment to ensure hyperphosphorylation and efficient elongation of Pol II.

Live‐cell imaging reveals the interplay between transcription factors, nucleosomes, and bursting

“…Recent studies have proposed a model where clustering or phase separation of the transcription machinery is important for driving transcription (Cisse et al , ; Hnisz et al , ; Lu et al , ). Although we do not exclude this possibility, it is important to note that the kinetics of our data can completely be described by the binding kinetics of individual TFs to single target genes, without need to invoke liquid droplets of multi‐molecular assemblies of multiple target genes.…”

Decoding the Notch signal

“…Furthermore, the transcription field has also seen a shift in paradigm. This "bursty" transcription has been formalized as a two state model of a promoter's activity, which cycles between ON and OFF states with stochastic rate constants (Larson, Singer, & Zenklusen, 2009;Peccoud & Ycart, 1995 The formation of PolII clusters or aggregates is thought to be driven by weak interactions between intrinsically disordered regions/low complexity domains (LCD) in PolII and other proteins (Boehning et al, 2018;Cho et al, 2018;Lu et al, 2018). This "bursty" transcription has been formalized as a two state model of a promoter's activity, which cycles between ON and OFF states with stochastic rate constants (Larson, Singer, & Zenklusen, 2009;Peccoud & Ycart, 1995 The formation of PolII clusters or aggregates is thought to be driven by weak interactions between intrinsically disordered regions/low complexity domains (LCD) in PolII and other proteins (Boehning et al, 2018;Cho et al, 2018;Lu et al, 2018).…”

RNAs, Phase Separation, and Membrane‐Less Organelles: Are Post‐Transcriptional Modifications Modulating Organelle Dynamics?

“…Generally, BioMCs have been conceptualized as RNP‐containing hubs in which complex biochemical reactions such as splicing, processing, and maturation of specific RNAs as well as metabolic turnover of various cellular components take place. However, particular BioMCs contribute also to specific biological processes such as polarity establishment and cellular identity in early embryos (i.e., P granules and RNP segregation in C. elegans ), asymmetric cell division of stem cells in Drosophila , transcription, heterochromatin formation, DNA repair, genome integrity through nuage‐affiliated processes, or the response to biotic and abiotic stress through the formation of SGs …”