Mechanisms of mRNA dihydrouridylation in Schizosaccharomyces pombe

Abstract

Modifications on noncoding- and coding-RNAs add an additional layer to the regulation of gene expression. Epitranscriptomics studies - focusing on mRNAs' modifications - are just in their beginnings. The study of the dihydrouridine (D) RNA modification, initiated in the lab by Olivier Finet and the development of the Rho-seq technique, revealed that D could be present within the coding region of 125 transcripts in fission yeast. In addition, genetic analyses support that the modification of mRNAs encoding cytoskeleton-associated proteins is required for the proper progression within meiosis and production of gametes. The dihydrouridylation of the nda2 transcript appears to be required for the production of the correct level of a-tubulin Nda2, which is essential for meiotic chromosomes segregation and in fine gametes viability. The dihydrouridine synthase 3 (Dus3) enzyme is a likely candidate to perform nda2 dihydrouridylation in vivo. Strains lacking dus3 are sterile and present similar phenotypes to a strain where the nda2 dihydrouridylation is abolished.
During this work, we pursued the investigation of the D regulatory functions. We successfully TAP tagged three of the four Dus enzymes in Schizosaccharomyces pombe, in order to purify them in the future. We began to investigate the mRNA dihydrouridylation molecular mechanism by conducting a RIP-qPCR analysis with the Dus3 enzyme in collaboration with the Rougemaille lab. In order to develop an orthogonal method to confirm the data obtained by D-seq, we set up serial poly-T purifications of mRNAs from WT and deleted Dus strains and performed a LC-MS/MS analyses of these fractions in collaboration with the Dedon lab. The preliminary data analysis is in favor with the presence of D in fission yeast mRNAs. We also focused on other putative dihydrouridylated transcripts beside nda2 including cct1, cct7 and zwf1 and used CRISPR to generate mutants of the putative target uridine. We successfully modified the sites where dihydrouridylation occurs. Our preliminary data did not reveal any apparent phenotypes though.
Further investigations must be pursued, especially during meiosis to further characterize Cct1, Cct7 and Zwf1 protein synthesis levels in addition to morphological analysis.

Date of Award	22 Jan 2021
Original language	English
Awarding Institution	University of Namur
Supervisor	Damien Hermand (Supervisor)