tRNA 2′-O-methylation by a duo of TRM7/FTSJ1 proteins modulates small RNA silencing 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 Nm methyltransferases (Nm-MTases) in Drosophila melanogaster (CG7009 and CG5220) as functional orthologs of yeast TRM7 and human FTSJ1. Genetic knockout studies together with MALDI-TOF mass spectrometry and RiboMethSeq mapping revealed that CG7009 is responsible for methylating the wobble position in tRNAPhe, tRNATrp and tRNALeu, while CG5220 methylates position C32 in the same tRNAs and also targets additional tRNAs. CG7009 or CG5220 mutant animals were viable and fertile but exhibited various phenotypes such as lifespan 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 catalyzing Nm at specific tRNAs and small RNA-induced gene silencing pathways.

tRNA Fragments Populations Analysis in Mutants Affecting tRNAs Processing and tRNA Methylation

“…In Drosophila it has been recently shown that methylation marks protect tRNAs from cleavage and that aberrant tRFs populations accumulate in methylation mutants: on the one hand, Dnmt2 mutation impairs m 5 C methylation (Schaefer et al, 2010;Durdevic et al, 2013a;Genenncher et al, 2018). On the other hand, dTrm7_34 (CG7009) and dTrm7_32 (CG5220) mutation impairs 2 -O-methylation (Angelova et al, 2020). 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 dTrm7_34 and dTrm7_32 are the functional orthologs of yeast TRM7 (Pintard, 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). 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 specific tRFs accumulation (Angelova et al, 2020).…”

“…Since tRFs biogenesis remains obscure, we developed and describe a user-friendly tRFs-pipeline for Drosophila melanogaster based on Galaxy environment (tRFs-Galaxy), with workflows and tools that can be easily shared with the scientific community. To do so, we took advantage of several Drosophila datasets (15-29 nt) generated in our laboratories: Rpp30 mutants, which affect tRNA processing, and dTrm7_34 and dTrm7_32, which affect tRNA Nm methylation (Molla-Herman et al, 2015;Mollà-Herman et al, 2019;Angelova et al, 2020). We believe that this study will help to better understand the known pathways of tRFs biogenesis as well as to uncover new tRFs biogenesis factors and unexpected crosstalks between different RNA regulatory mechanisms, crucial for gene expression.…”

“…RNA was extracted from Drosophila ovaries following standard methods detailed in Molla-Herman et al 2015and Angelova et al (2020).…”

“…Workflows are available upon request. Data set deposition is described in Molla-Herman et al 2015and Angelova et al (2020). European Nucleotide Archive (ENA) of the EMBL-EBI 6 , accession numbers are: PRJEB10569 (Rpp30 mutants), PRJEB35301 and PRJEB35713 (Nm mutants).…”