Research
RNA polymerase II CTD and transcriptional elongation checkpoints
RNA Polymerase II (pol II) is a key player in the coupling of transcription and mRNA co-transcriptional processes such as mRNA capping and splicing. Pol II largest subunit has a very unusual C-terminus structure comprised of 52 repeats in human of the heptapeptide Tyr1Ser2Pro3Thr4Ser5Pro6Ser7 (YSPTSPS). This structure is called the carboxyl-terminal domain, or pol II CTD, and undergoes post-translational modifications like phosphorylation, acetylation or methylation.
A complex interplay of enzymes that either add or remove modifications results in a dynamic range of CTD modifications during the transcription cycle. This CTD "code" is known to orchestrate the sequential recruitment of transcription and RNA processing factors. Good progress has been made towards a extensive understanding of how this code is written and read.
One of the enzymes involved in phosphorylating the Ser2 (S2) residue of the pol II CTD is P-TEFb, which is comprised of a kinase, CDK9, and a cyclin, Cyclin T1 or cyclin T2. Phosphorylation by P-TEFb of the pol II CTD and two other proteins, Spt5 and NELF-E, is required at the start of genes for the transition of pol II to productive elongation, a checkpoint called the early-elongation checkpoint. Using inhibitors of the CDK9 kinase subunit of P-TEFb, we uncovered a hitherto-unsuspected kinase-dependent checkpoint close to the poly(A) site. This second checkpoint, named the poly(A)-associated 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. We are currently investigating the molecular mechanism underlying this novel transcription elongation checkpoint by producing with CRISPR/Cas9 analog-sensititve kinases, allowing us to inhibit specifically a single kinase, and determining their functions using genome-wide nascent RNA-sequencing (mNET-seq) and phosphoproteomics.
Laitem, C., Zaborowska, J., Isa, N.F., Kufs, J., Dienstbier, M. and Murphy, S., 2015. CDK9 inhibitors define elongation checkpoints at both ends of RNA polymerase II–transcribed genes. Nature structural & molecular biology, 22(5), pp.396-403.
Zaborowska, J., Egloff, S. and Murphy, S., 2016. The pol II CTD: new twists in the tail. Nature Structural & Molecular Biology, 23(9), pp.771-777.
Tellier, M., Ferrer-Vicens, I. and Murphy, S., 2016. The point of no return: The poly (A)-associated elongation checkpoint. RNA biology, 13(3), pp.265-271.
Transcriptional regulation of snRNA genes
The small non-coding RNAs (snRNAs) are short, intronless and non-polyadenylated genes involved in splicing, rRNA processing and 3’end histones formation. The snRNA gene structure differs from protein coding genes. It comprises an enhancer distal sequence element (DSE), a promoter sequence element (PSE) and a 3’box which is responsible for RNA 3’end formation. Previous studies have shown a coupling between DSE/PSE and 3’box. Indeed, when the PSE is swapped for protein coding gene promoter, the 3’box signal is not recognized anymore and pol II uses a downstream polyadenylation site. Although there are some factors known to be involved exclusively on the snRNA genes regulation, it is still unclear how this coupling happened. We are currently using an unbiased approach to identify factors involved in snRNA gene transcription that could fully elucidate how those genes are regulated.
Guiro, J., & Murphy, S. (2017). Regulation of expression of human RNA polymerase II-transcribed snRNA genes. Open biology, 7(6), 170073.