TRNA 2'-O-methylation by a duo of TRM7/FTSJ1 proteins modulates small RNA silencing in Drosophila.

Mira Brazane,Margarita Angelova ,Christophe Antoniewski,Virginie Marchand,Cyrinne Achour,Dilyana G Dimitrova,Caroline Jacquier,Souraya Khouider,Salman Shehzada,Clément Carré,Laure Teysset,Yuri Motorin,Catherine Goyenvalle,Damien Brégeon ,Bruno Da Silva ,Vincent Guérineau ,Matthias Schaefer ,Valérie Bourguignon‐Igel ,Tina Lenče ,Jean‐Yves Roignant

doi:10.1093/nar/gkaa002

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.

Highlights

The existence of RNA modifications has been known for over 50 years and many of the pioneering studies addressed the function of RNA modifications in abundantly expressed RNAs such as transfer RNAs and ribosomal RNAs. tRNAs are the most heavily modified RNAs. tRNAs are modified post-transcriptionally and the biosynthesis of modified nucleosides requires different modification enzymes acting sometimes sequentially at distinct steps of tRNA maturation [2,3]
While performing an RNAi genome-wide screen for modulators of miRNA biogenesis or Ago2-dependent small RNA silencing in Drosophila, we identified a previously uncharacterized gene (CG7009) with sequence homology to Nm-MTases of the TRM7/FTSJ1 subfamily of MTase proteins
When re-testing the automiG read-out (GFP expression) upon efficient CG5220 knockdown in S2 cells, we obtained variable and inconclusive results, which supported the fact that CG5220 was not uncovered as modulator of miRNA biogenesis or Ago2-dependent small RNA silencing by the original screen in S2 cells

Summary

Introduction

The existence of RNA modifications has been known for over 50 years and many of the pioneering studies addressed the function of RNA modifications in abundantly expressed RNAs such as transfer RNAs (tRNAs) and ribosomal RNAs (rRNAs). tRNAs are the most heavily modified RNAs (up to 25% nucleotides/tRNA, [1]). tRNAs are modified post-transcriptionally and the biosynthesis of modified nucleosides requires different modification enzymes acting sometimes sequentially at distinct steps of tRNA maturation [2,3]. TRNAs are modified post-transcriptionally and the biosynthesis of modified nucleosides requires different modification enzymes acting sometimes sequentially at distinct steps of tRNA maturation [2,3]. The complex mechanisms underlying the stepwise modification of tRNAs were largely deciphered in the yeast Saccharomyces cerevisiae, as well as in studies conducted in prokaryotes and Archaea. Nucleic Acids Research, 2020, Vol 48, No 4 2051 at understanding how mutations in tRNA modification enzymes affect organismal development and disease etiology

Methods

Results

Conclusion