Role of the RNA Polymerase II CTD-S2 phosphorylation by CDK-12 during the post-embryonic development in the nematode C. elegans

Abstract

The C-terminal domain (CTD) of the RNA Polymerase II is composed of repeats of the consensus sequence Y1S2P3T4S5P6S7. The CTD is highly modified during transcription, which plays a major role in the coordination of transcription and mRNA maturation. CDK-12 phosphorylates the CTD-S2, a mark classically associated with elongation, splicing and cleavage/polyadenylation.
However, recent studies in yeast, fly and mammalian cell, highlighted a gene-specific requirement for CDK-12. For instance, in fission yeast, the CTD-S2 phosphorylation is only required for the induction of sexual differentiation.
In C. elegans, cdk-12 knockdown by RNAi results in a L1 arrest. The embryos hatch in presence of food but are unable to induce the post-L1 development, mimicking a L1 arrest in starvation. Homozygous cdk-12 disruption leads to the same terminal phenotype.
We have constructed an analog-sensitive (as) version of CDK-12, cdk-12as, to investigate the role of the CTD-S2 phosphorylation by CDK-12 during the early larval development. In the presence of low dose of the ATP analog, the cdk-12as strain arrests at the L1 stage with undetectable CTD-S2 phosphorylation. We show that the inhibition is specific, very fast, and can be reversed by washing out the ATP analogue.
Transcriptomic analyses by RNA-seq were performed during physiological or ATP analog-induced L1 arrest, which revealed that the loss of CDK-12 kinase activity only affects a subset of genes strongly enriched for “development related genes”. Deeper analyses indicated that most target genes belong to operons and undergo SL2 trans-splicing. Therefore, our results suggest that the CDK-12 kinase is specifically required for the induction of SL2-trans-spliced development related genes.
In addition, our data indicate that the RNA Polymerase II occupancy along the operons is barely affected when CDK-12 is inhibited, suggesting a posttranscriptional requirement, maybe during trans-splicing.
Taken together our results show that the phosphorylation of CTD-S2 by CDK-12 is dispensable during embryogenesis but is required to escape L1 arrest and pursue larval development.

Date of Award	17 Mar 2017
Original language	English
Awarding Institution	University of Namur
Sponsors	Fund for Research Training in Industry and Agriculture (FRIA)
Supervisor	Damien Hermand (Supervisor), Olivier De Backer (President), Valérie Robert (Jury), Patrick Laurent (Jury) & René Rezsöhazy (Jury)