During transcription of both protein-coding and snRNA genes by RNA polymerase II (pol II), transcription and RNA processing are tightly coupled. Our most recent work has focused on understanding the mechanics of this connection and the role of the transcriptional cyclin-dependent kinases in this process (1-8).
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EC-elongation complex TC-termination complex. The size of the circled P (phosphate) reflects the relative level of phosphorylation.
Pol II itself is a key player in this coupling and can profoundly influence how factors are recruited for transcription and RNA processing. The largest subunit of pol II has a very unusual structure at the C-terminus called the carboxyl-terminal domain or CTD, with 52 repeats of the consensus heptapeptide tyrosine/serine/proline/threonine/serine/proline/serine or Y1/S2/P3/T4/S5/P6/S7 for short. Intriguingly, each of the amino acids can be modified; tyrosine, serine and threonine by phosphorylation and proline by isomerisation and all modifications have been identified in living cells (1). There is, therefore, the potential for a very large number of different combinations of modifications along the CTD.
CTD modifications are known to orchestrate the sequential recruitment of transcription and RNA processing factors during the transcription cycle and a complex interplay of enzymes that either add or remove modifications results in changes in CTD modification during the transcription cycle. For example, phosphorylation of S5 predominates early in transcription of both protein-coding and snRNA genes, while phosphorylation of S2 predominates towards the end. It has been suggested that the many different potential combinations of CTD modifications produces a code that is read by the factors recruited for transcription and RNA processing. Good progress is being made towards a full understanding of how this code is written and read and interestingly, some CTD modifications appear to play a specific role in expression of snRNA genes (7, 8).
One of the enzymes involved in phosphorylation of S2 of the pol II CTD, P-TEFb, also controls the ability of pol II to pass an early-elongation checkpoint. Using inhibitors of the CDK9 kinase subunit of P-TEFb, we have mapped early-elongation checkpoints on pol II-transcribed genes genome-wide. Surprisingly, this analysis also uncovered a hitherto-unsuspected kinase-dependent checkpoint close to the poly(A) site. This second checkpoint could provide the opportunity to rapidly regulate gene expression by terminating pol II just before the production of a polyadenylated mRNA; the point of no return. We have already shown that CTD phosphorylation changes and some polyadenylation/elongation factors are lost from the region of the poly(A) site when cells are treated with CDK9 inhibitors (Image 1) (2,7). We are currently investigating the molecular mechanism underlying this novel transcription elongation checkpoint.
We also study the mechanics of expression of the small nuclear (sn)RNA genes transcribed by pol II (6), whose non-coding RNA products are involved in processing other RNAs. For example several snRNA are required for the process of splicing.
2022
CDK9 and PP2A regulate RNA polymerase II transcription termination and coupled RNA maturation.
Tellier, M., Zaborowska, J., Neve, J., Nojima, T., Hester, S., Fournier, M., Furger, A. and Murphy, S.
EMBO Rep. – 23(10): e54520.
2022
CAPTURE of the Human U2 snRNA Genes Expands the Repertoire of Associated Factors.
Guiro, J., Fagbemi, M., Tellier, M., Zaborowska, J., Barker, S., Fournier, M. and Murphy, S.
Biomolecules – 12(5): 704.
2020
CDK12 globally stimulates RNA polymerase II transcription elongation and carboxyl-terminal domain phosphorylation.
Tellier, M., Zaborowska, J., Caizzi, L., Mohammad, E., Velychko, T., Schwalb, B., Ferrer-Vicens, I., Blears, D., Nojima, T., Cramer, P. and Murphy, S.
Nucleic Acids Research – 48(14): 7712-7727.
2018
Deregulated Expression of Mammalian lncRNA through Loss of SPT6 Induces R-Loop Formation, Replication Stress, and Cellular Senescence.
Nojima, T., Tellier, M., Foxwell, J., Ribeiro de Almeida, C., Tan-Wong, S.M., Dhir, S., Dujardin, G., Dhir, A., Murphy, S. and Proudfoot, N.J.
Molecular Cell – 72(6): 970-984.e7.
2017
Regulation of expression of human RNA polymerase II-transcribed snRNA genes.
Guiro, J. and Murphy, S.
Open Biol. – 7(6): 170073.
2016
The pol II CTD: new twists in the tail.
Zaborowska, J., Egloff, S. and Murphy, S.
Nat Struct Mol Biol. – 23(9): 771-7.
2015
CDK9 inhibitors define elongation checkpoints at both ends of RNA polymerase II-transcribed genes.
Laitem. C., Zaborowska, J., Isa, N.F., Kufs, J., Dienstbier, M. and Murphy, S.
Nat Struct Mol Biol. – 22(5): 396-403.
2007
Serine-7 of the RNA polymerase II CTD is specifically required for snRNA gene expression.
Egloff, S., O’Reilly, D., Chapman, R.D., Taylor, A., Tanzhaus, K., Pitts, L., Eick, D. and Murphy, S.
Science – 318(5857): 1777-9.