AbstractTranscription of protein coding genes is carried out by the RNA polymerase II (Pol II). It is now clearly established that Pol II itself is subject to many modifications that can profoundly influence how factors required for transcription and RNA processing are recruited. The largest subunit of Pol II harbors a tail like C-terminal domain (CTD) composed of repeats of the consensus heptapeptide sequence Y1S2P3T4S5P6S7. It has been proposed that many different potential combinations of CTD modifications makes up a code that orchestrate the complex sequential recruitments of factors involved in transcription and RNA processing. One of the most abundant CTD modification is the phosphorylation of the CTD-Ser2, a mark classically associated with transcription elongation. The positive elongation complex CDK9/Cyclin T(P-TEFb) has been shown to phosphorylate CTD-Ser2 and to be broadly essential for expression of early embryonic genes.
In the model organism Caenorhabditis elegans, the RNAi knock down of cdk-9 mRNA induces an early embryonic arrest mimicking an RNAi knock down of ama-1, the biggest Pol II subunit. We have constructed a cdk-9 analogue sensitive (cdk-9as) strain seeking to investigate the role of CDK-9 during development. We showed that the inhibition of CDK-9as with the bulky ATP analogue 3MB-PP1 does not perfectly phenocopy RNAi knock down but induces a reduction in brood size and partial embryonic lethality. It has been proposed that CDK-12, but not CDK-9, is the major CTD-Ser2 kinase in C. elegans. Using our newly constructed cdk-9as mutant strain, we investigated the relative contribution of CDK-9 in the phosphorylation of CTD-Ser2. Surprisingly, we did not detect a drop in the phosphorylation level upon CDK-9as inhibition. CDK-12as inhibition causes a severe decrease in CTD-Ser2P but detectable signal is still present. We constructed a mutant strain containing both cdk-9as and cdk-12as and showed that simultaneous kinase inhibition at high concentration causes a more severe drop in CTD-Ser2 phosphorylation level than CDK-12as inhibition alone. We postulate that the CTD kinase activity of CDK-9 and CDK-12 may be partially redundant. When CDK-9as is inhibited, CDK-12 alone can compensate for the loss in CTD-Ser2 phosphorylation. Taken together our results show that CDK-9 dependent CTD-Ser2 phosphorylation is required for completion of embryogenesis but the CDK-9 itself is dispensable to maintain proper level of CTD-Ser2 phosphorylation.
|Date of Award||18 Jan 2019|
|Supervisor||Damien Hermand (Supervisor)|
- C. elegans