High-resolution mapping of elongating RNA polymerase II (RNAPII) using Native Elongating Transcript sequencing (NET-seq) revealed frequent pausing events in the 5’end of genes (Churchman and Weissman, 2011). More recently, we found that these early elongation pausing events are reduced dramatically when the RNAPII C-terminal domain (CTD) is mutated to replace its tyrosines with phenylalanines (Collin et al., 2019). Because the CTD is well known to recruit different factors to the elongating RNAPII, we then performed a proteomic analysis of this RNAPII-Y1F mutant but found no tyrosine-dependent RNAPII interactor likely to provide an explanation to the loss of pausing phenotype. Mounting evidence suggests that the CTD can form protein condensates in cells via liquid-liquid phase separation (Boehning et al., 2018; Guo et al., 2019; Lu et al., 2018). Protein condensates formed by transcription factors, co-activators, and RNAPII are major players in gene expression regulation, notably by providing local environments favorable for the assembly of macromolecular assemblies such as super-enhancers (Boija et al., 2018; Cho et al., 2018; Sabari et al., 2018). Interestingly, CTD phosphorylation, which is a very dynamic process during transcription elongation (Jeronimo et al., 2013), was shown to affect the ability of the CTD to form condensates (Boehning et al., 2018) and to drive RNAPII from one type of condensate to another (Guo et al., 2019; Lu et al., 2018). Based on these data, and because tyrosine residues are often determinant for the ability of a protein to form condensates (Wang et al., 2018), we hypothesize that 1) the transition from initiation to elongation involves a redistribution of RNAPII from initiation-type condensates into elongation-type condensates; 2) this transition is a rate-limiting step resulting in pausing and 3) the phosphorylation of the CTD provides the driving force for this transition. Base on this model, we surmise that the RNAPII-Y1F does not enter initiation condensates, hence bypassing the rate-limiting transition from initiation to elongation condensates. We, therefore, propose to use the RNAPII-Y1F mutant as a tool to test the model described above.
1) Test the ability of recombinant CTD variants (WT, Y1F, and others) to form liquid droplets in vitro and to appear as puncta in cells.
2) Fuse the Y1F CTD with protein domains know to drive the formation of protein condensates and test whether it restores the ability of the Y1F CTD to phase separate in vitro and form puncta in cells. IDRs that may work here are those known to form condensate with RNAPII and Mediator (e.g. IDRs from OCT4, MYC, p53, NANOG, SOX2, BRD4, β-catenin, STAT3, SMAD3, etc.). On the other hands IDRs that can phase separate but not with RNAPII and enhancers would be predicted to fail in this assay (e.g. splicing factors IDRs or heterochromatin protein IDRs)
3) Test whether these fusions restore pausing in vivo by NET-seq.
Boehning, M., Dugast-Darzacq, C., Rankovic, M., Hansen, A.S., Yu, T., Marie-Nelly, H., McSwiggen, D.T., Kokic, G., Dailey, G.M., Cramer, P., et al. (2018). RNA polymerase II clustering through carboxy-terminal domain phase separation. Nat Struct Mol Biol 25, 833-840.
Boija, A., Klein, I.A., Sabari, B.R., Dall’Agnese, A., Coffey, E.L., Zamudio, A.V., Li, C.H., Shrinivas, K., Manteiga, J.C., Hannett, N.M., et al. (2018). Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains. Cell 175, 1842-1855 e1816.
Cho, W.K., Spille, J.H., Hecht, M., Lee, C., Li, C., Grube, V., and Cisse, II (2018). Mediator and RNA polymerase II clusters associate in transcription-dependent condensates. Science 361, 412-415.
Churchman, L.S., and Weissman, J.S. (2011). Nascent transcript sequencing visualizes transcription at nucleotide resolution. Nature 469, 368-373.
Collin, P., Jeronimo, C., Poitras, C., and Robert, F. (2019). RNA Polymerase II CTD Tyrosine 1 Is Required for Efficient Termination by the Nrd1-Nab3-Sen1 Pathway. Mol Cell 73, 655-669 e657.
Guo, Y.E., Manteiga, J.C., Henninger, J.E., Sabari, B.R., Dall’Agnese, A., Hannett, N.M., Spille, J.H., Afeyan, L.K., Zamudio, A.V., Shrinivas, K., et al. (2019). Pol II phosphorylation regulates a switch between transcriptional and splicing condensates. Nature 572, 543-548.
Jeronimo, C., Bataille, A.R., and Robert, F. (2013). The writers, readers, and functions of the RNA polymerase II C-terminal domain code. Chem Rev 113, 8491-8522.
Lu, H., Yu, D., Hansen, A.S., Ganguly, S., Liu, R., Heckert, A., Darzacq, X., and Zhou, Q. (2018). Phase-separation mechanism for C-terminal hyperphosphorylation of RNA polymerase II. Nature 558, 318-323.
Sabari, B.R., Dall’Agnese, A., Boija, A., Klein, I.A., Coffey, E.L., Shrinivas, K., Abraham, B.J., Hannett, N.M., Zamudio, A.V., Manteiga, J.C., et al. (2018). Coactivator condensation at super-enhancers links phase separation and gene control. Science 361.
Wang, J., Choi, J.M., Holehouse, A.S., Lee, H.O., Zhang, X., Jahnel, M., Maharana, S., Lemaitre, R., Pozniakovsky, A., Drechsel, D., et al. (2018). A Molecular Grammar Governing the Driving Forces for Phase Separation of Prion-like RNA Binding Proteins. Cell 174, 688-699 e616.
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