Abstract
The transcription of protein-coding genes is realized by the RNA polymerase II (Pol II). This enzyme is composed of 12 subunits named Rpb1 to Rpb12 and possesses on its largest subunit, Rpb1, a tail-like carboxy-terminal domain (CTD) composed of repetitions of the heptapeptide sequence Y1-S2-P3-T4-S5-P6-S7. Each of the CTD residues can be post-translationally modified to form different combinations allowing factors involved in mRNA processing and maturation to dock on Pol II. The most abundant modifications are the phosphorylation of the serine in position 2 (CTD-Ser2) and 5 (CTD-Ser5) by the Cyclin-Dependent Kinases, CDK7 (which phosphorylates the CTD-Ser5) and CDK9/CDK12 (which phosphorylate the CTD-Ser2). Although CDK9 is essential for embryogenesis, CDK12 is not and is rather required for the expression of a subset of genes like genes involved in postembryonic development in the model organism Caenorhabditis elegans. Indeed, previous work from the lab showed, that the inactivation of CDK-12 in C. elegans results in an early post-embryonic developmental arrest at the L1 stage.The goal of this master thesis is to pursue the study of CDK-12 by checking that the phenotype induced by its inactivation is indeed consequent to a deficit of CTD-Ser2 phosphorylation and second, by studying how CDK-12 is regulated in vivo. We thus aimed to create two C. elegans mutant strains, one expressing a CTD where the CTD-Ser2 are replaced by a non-phosphorylable residue and another expressing a tagged CDK-12 to study its phosphorylation status and putative partners by mass spectrometry. To generate these two strains, we used several variations of the CRISPR/Cas9 method, so far with no success. A transgenic control strain targeting another gene was obtained, which indicates that the method is efficient.
| Date of Award | 17 Jan 2020 |
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| Original language | English |
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| Supervisor | Damien Hermand (Supervisor) |