N6-methyladenosine, a new modification in T. brucei epitranscriptome

Abstract

Trypanosoma bruceiis a unicellulareukaryote parasite that causes human sleeping sickness. In this parasite, transcription is mainly constitutive and gene expression regulation occurs essentially at post-transcriptional level. N6-methyladenosine (m6A) is an RNA modification associated withpost-transcriptional gene regulation in eukaryotes. These observations led to the proposalthat this modification occurs in T. bruceitranscriptome and is involved in post-transcriptional gene regulation. In this thesis, m6A was detected for the first time in T. bruceiRNA and additionally in DNA, from bloodstream and procyclic life stages. As faras Iknow, this is the first description of an organism in which has m6A is found in both type of nucleic acids. In RNA, I observed that the levels are regulatedin different biological circumstances, namely, it increases during differentiation from bloodstream to procyclic life-cycle stages and it also increases when parasites are stressed by being placed at high cell density. T. bruceigenome was searched with bioinformatics tools to find enzymes that catalysethe formation and the removal the of m6A modification in RNA. One putative RNA m6A methyltransferase and six putative demethylases were found. Knockout cell lines of the putative methyltransferase (Tb927.7.6620)and of two putative demethylases (Tb927.4.460 named TbALKBH1 and Tb927.5.980 named TbALKBH2) were generated to test their putative functions. Quantification of m6A levels in RNA from the knockout cell lines did not reveal evidence that support the putative function of the methyltransferaseand one demethylase (TbALKBH1). However, knockout of TbALKBH2 resulted in a slight increase in m6A levels, suggesting that this candidate could be an RNA m6A demethylase. The evidence presented in this thesis raises the possibilityof post-transcriptional gene regulation mediated by the presence of m6A modification in T. bruceiepitranscriptome.