CDK9 and PP2A regulate the link between RNA polymerase II transcription termination and RNA maturation

Michael Tellier, Justyna Zaborowska, Jonathan Neve, Takayuki Nojima, Svenja Hester, Andre Furger, Shona Murphy

Preprint posted on 21 June 2021 https://www.biorxiv.org/content/10.1101/2021.06.21.449289v1

Article now published in EMBO reports at http://dx.doi.org/10.15252/embr.202154520

CDK9 and PP2A counteract each other and are key in the regulation of RNA polymerase II transcription and in the coordination of RNA processing with transcription.

Selected by Isabella Maudlin

Categories: biochemistry, bioinformatics, cell biology, genomics, molecular biology

Background

RNA polymerase II transcribes genes encoding all proteins (mRNAs), micro RNAs, small nuclear RNAs and small nucleolar RNAs. From yeast to humans, RNA polymerase II has a large unstructured C-terminal domain called the CTD which consists of a variable number of repeats with the consensus sequence YSPTSPS. During transcription, the CTD is extensively post-translationally modified according to its transcriptional progress along a gene. The CTD has been likened to a ‘landing pad’ for the recruitment of transcription elongation factors that regulate stages of RNAPII transcription and for the recruitment of RNA processing factors during transcription.

The most well-studied post-translational modification of the CTD is phosphorylation – 5 of the 7 residues can be reversibly phosphorylated by CTD kinases which are recruited at the appropriate stage during transcription. One such CTD kinase is CDK9, which is critical for RNA polymerase II transcription in higher eukaryotes. Without CDK9 activity, RNA polymerase II does not escape the promoter and transcription cannot occur. Alternatively, RNA polymerase II present in the gene prematurely terminates close to the polyadenylation site, resulting in the loss of polyadenylation factors and defects in transcript polyadenylation. Though likely to also play key roles in transcription, the role of phosphatases is less clear.

In this preprint, Tellier et al. used a chemical genetics approach, in which CDK9 is mutated to accept “bulky” ATP-analogs that rapidly inhibit kinase activity, to study the function of CDK9 during transcription. Further, the authors discover a novel role for phosphatase PP2A in counteracting CDK9 activity, and demonstrate that the activities of CDK9 and PP2A are key not only in the regulation of RNA polymerase II transcription, but in the coordination of RNA processing with transcription.

Key Findings

1. CDK9 inhibition causes loss of polyadenylation and causes a transcription defect in the last exon of genes

To understand kinetics of transcription after CDK9 inhibition, the authors carried out a time course of mammalian native elongating transcript-sequencing (mNET-SEQ). mNET-SEQ involves a native pull-down of engaged RNAPII followed by purification of associated nascent RNA transcripts and sequencing the 3’ ends, allowing the mapping of RNA polymerase II during transcription to base-pair resolution. As expected, CDK9 inhibition caused an accumulation of RNA polymerase II at the promoter, a consequential loss of RNA polymerase II in gene bodies, and premature termination close to the polyadenylation site. Interestingly, further analysis revealed that RNA polymerase II transcription is affected from the last exon, not from the polyadenylation site as previously thought, and that RNA polymerase II is lost past the polyadenylation site. In addition, inhibition of CDK9 caused dissociation of polyadenylation and termination factors from chromatin, and reduced polyadenylation of transcripts.

2. CDK9 phosphorylates transcription and splicing factors

To identify in vivo targets of CDK9, phosphoproteomics were performed with or without inhibition of CDK9, and the phosphorylation states compared. 100 phosphosites were found to decrease by more than 1.5-fold across 74 different proteins after CDK9 inhibition. Known targets of CDK9 were identified, including transcription elongation factor SPT5 and splicing factor SF3B1. Interestingly, novel phosphosites on each of these proteins were identified and validated by the generation of antibodies. The phosphorylation of SF3B1 had been previously shown to be required for splicing and for chromatin-association of the spliceosome. Tellier et al. further these works by finding that CDK9 inhibition reduces the association of key splicing factors and RNA export factors with SF3B1. Further, they find that CDK9 inhibition affects co-transcriptional splicing.

3. PP2A counteracts CDK9 activity at the 3’ ends of genes and together CDK9 and PP2A regulate transcript cleavage and polyadenylation

To identify which kinase might counteract CDK9 activity, PP1 and PP2A were inhibited at the same time as CDK9, and the effects on transcription analysed. Whilst inhibition of PP1 and CDK9 caused similar transcription defects as observed with CDK9 inhibition alone, inhibition of PP2A and CDK9 prevented the premature termination of RNA polymerase at the 3’ ends of genes. CDK9 inhibition also causes loss of transcript cleavage and polyadenylation. To test the role of PP2A in this phenomenon, CDK9 and PP2A were inhibited at the same time. This prevented the loss of polyadenylation caused by CDK9 inhibition. Interestingly, PP2A inhibition alone caused an increase in polyadenylation, suggesting that PP2A normally acts as a negative regulator of this process.

Together, these data show that PP2A, but not PP1, counteracts CDK9 activity at the 3’ ends of genes and prevents the premature termination and loss of transcript cleavage and polyadenylation associated with CDK9 inhibition.

What I like about this preprint

Whilst a lot is known about the regulation of transcription and co-transcriptional RNA processing by CTD kinases, little is known about the function of phosphatases and their counteracting of CTD kinase activity. This study uses high-throughput techniques to show that not only do we still have more to learn about CTD kinases, but that the phosphatases are key players too.

Posted on: 2 November 2021

doi: https://doi.org/10.1242/prelights.30946

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Author's response

Michael Tellier shared

Do you think that the loss of SF3B1 phosphorylation on its own causes the splicing defects observed, or do you think it is a combination of splicing factors losing phosphorylation?

I think it is a result of phosphorylation changes across several splicing factors . In our phosphoproteomics experiment, we found that CDK9 phosphorylates on (S/T)P sites SF3B1 and CDC5L but there is also non (S/T)P sites that are dephosphorylated in other splicing factors, in HTATSF1 or hnRNPA1 for example. In addition, some other splicing factors, such as SRRM2 or SRSF10, have a higher phosphorylation level following CDK9 inhibition and could therefore affect pre-mRNA splicing. Further work is required, especially by looking at the function(s) of single phosphorylation sites, to understand better the role of CDK9 kinase activity in pre-mRNA splicing. Another important question will be to determine if CDK9 regulates pre-mRNA splicing globally or if it plays a more restricted role.

Do you know if PP2A affects phosphorylation of splicing factors and splicing?

A 2020 paper from Jukka Westermarck’s group in the Journal of Biological Chemistry has shown that PP2A can dephosphorylate several splicing factors, including SRSF3 and different hnRNP proteins. In addition, James Manley’s group in 2006 has shown in vitro that PP1 and PP2A were playing essential but redundant roles in splicing, especially at the second step of splicing. In our experiments, we have not observed a splicing defect following PP1 or PP2A inhibition, even though we know that for example SF3B1 phosphorylation is affected by PP1 inhibition. This absence of a splicing defect following PP1 or PP2A inhibition might be due to the potential redundancy of these two phosphatases, as proposed by James Manley’s group. It is something we are currently investigating further.

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CDK9 and PP2A regulate the link between RNA polymerase II transcription termination and RNA maturation

Background

Key Findings

1. CDK9 inhibition causes loss of polyadenylation and causes a transcription defect in the last exon of genes

2. CDK9 phosphorylates transcription and splicing factors

3. PP2A counteracts CDK9 activity at the 3’ ends of genes and together CDK9 and PP2A regulate transcript cleavage and polyadenylation

What I like about this preprint

Share this:

Have your say Cancel reply

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