Etude du rôle de la kinase Cdk-12 dans le développement post-embryonnaire chez le nématode C. elegans

Promoter

Dr. Damien HERMAND, UNamur, Molecular Physiology Research Unit (URPhyM), Molecular Genetics (GeMo)

Jury

Jury extérieur : Dr. Patrick LAURENT, ULB Campus Erasme, Bruxelles ; Dr. René REZSOHAZY, UCL, ISV, Louvain la Neuve ; Dr. Valérie ROBERT, Ecole Normale, Lyon, France.

Jury intérieur : Prof. Olivier DE BACKER, Dpt. Sciences Biomédicales, Faculté de Médecine ; Prof. Damien HERMAND, Dpt. Sciences Biomédicales, Chercheur FNRS, Faculté de Médecine.

Summary

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.