tRNA 2’-O-methylation modulates small RNA silencing and life span in Drosophila

Abstract

2'-O-methylation (Nm) represents one of the most common RNA modifications. Nm affects RNA structure and function with crucial roles in various RNA-mediated processes ranging from RNA silencing, translation, self versus non-self recognition to viral defense mechanisms. Here, we identify two novel Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1, respectively. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNA Phe , tRNA Trp and tRNA Leu , while subsequently, CG5220 methylates position C32 in the same tRNAs and targets also additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as life span reduction, small RNA pathways dysfunction and increased sensitivity to RNA virus infections. Our results provide the first detailed characterization of two TRM7 family members in Drosophila and uncover a molecular link between enzymes catalysing Nm at specific tRNAs and small RNA-induced gene silencing pathways.

tRNA fragments (tRFs) populations analysis in mutants affecting tRNAs processing and tRNA methylation

“…RNA was extracted from Drosophila ovaries following standard methods detailed in (Molla-Herman et al, 2015;M. Angelova, 2019).…”

“…In Drosophila it has been recently shown that methylation marks protect tRNAs from cleavage and aberrant tRFs populations accumulate in mutants: on the one hand, Dnmt2 mutation impairs m 5 C methylation (Schaefer et al, 2014;Genenncher et al, 2018) and on the other hand, CG7009 and CG5220 mutation impairs 2'-O-methylation (M. Angelova, 2019). 2'-O-methylation is one of the most common RNA modifications and consists in the addition of a methyl group to the 2' hydroxyl of the ribose moiety of a nucleoside, being also known as Nm.…”

“…It has been recently shown that Drosophila proteins CG7009 and CG5220 are the functional orthologues of yeast TRM7 (Pintard et al, 2002) and human FTSJ1 (Guy et al, 2015) respectively, which are involved in 2'-O-methylation of the anticodon loop of several conserved tRNAs substrates (tRNA-Leu, Trp, Phe) in different species, including Drosophila. Indeed, mutations of these tRNAs methyltransferases in Drosophila lead to lifespan reduction, small non-coding RNA pathways dysfunction and increased sensitivity to RNA virus infections, besides tRFs accumulation (M. Angelova, 2019).…”

“…Since tRFs biogenesis remains obscure, we developed and described a "user-friendly" pipeline based on Galaxy environment to study all types of tRFs, including those that have a CCA tag. To do so, we took advantage of several Drosophila datasets generated in our laboratories: Rpp30 mutants, which affect tRNA processing, and CG7009 and CG5220, which affect tRNA methylation (Molla-Herman et al, 2015;M. Angelova, 2019).…”

“…All the analyses were performed with Galaxy tools https://mississippi.snv.jussieu.fr. Data set deposition is described in (Molla-Herman et al, 2015;M. Angelova, 2019).…”