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

Anahi Molla-Herman,Christophe Antoniewski,Christophe Antoniewski,Christophe Antoniewski,Jean-René Huynh,Jean-René Huynh,Jean-René Huynh,Jean-René Huynh,Margarita T Angelova,Margarita T Angelova,Margarita T Angelova,Margarita T Angelova,Margarita T Angelova,Clément Carré,Clément Carré,Clément Carré,Clément Carré,Clément Carré,Maud Ginestet,Maud Ginestet,Maud Ginestet,Maud Ginestet

doi:10.3389/fgene.2020.518949

Abstract

tRNA fragments (tRFs) are a class of small non-coding RNAs (sncRNAs) derived from tRNAs. tRFs are highly abundant in many cell types including stem cells and cancer cells, and are found in all domains of life. Beyond translation control, tRFs have several functions ranging from transposon silencing to cell proliferation control. However, the analysis of tRFs presents specific challenges and their biogenesis is not well understood. They are very heterogeneous and highly modified by numerous post-transcriptional modifications. Here we describe a bioinformatic pipeline (tRFs-Galaxy) to study tRFs populations and shed light onto tRNA fragments biogenesis in Drosophila melanogaster. Indeed, we used small RNAs Illumina sequencing datasets extracted from wild type and mutant ovaries affecting two different highly conserved steps of tRNA biogenesis: 5′pre-tRNA processing (RNase-P subunit Rpp30) and tRNA 2′-O-methylation (dTrm7_34 and dTrm7_32). Using our pipeline, we show how defects in tRNA biogenesis affect nuclear and mitochondrial tRFs populations and other small non-coding RNAs biogenesis, such as small nucleolar RNAs (snoRNAs). This tRF analysis workflow will advance the current understanding of tRFs biogenesis, which is crucial to better comprehend tRFs roles and their implication in human pathology.

Highlights

Transfer RNAs are molecules of ∼75 nt transcribed by RNA polymerase III that adopt a typical cloverleaf secondary structure
In this study we have developed user friendly and easy to share workflows using Galaxy5 allowing to extract all major classes of Transfer RNAs (tRNAs) fragments (tRFs) (Figure 1 and see section “MATERIALS AND METHODS”): 5 -tRFs, 3 -tRFs and inner-tRFs, corresponding to fragments derived from mature tRNA transcripts; tRFs-1, formed by RNase-Z cleavage of tRNA precursors; spanner tRFs, spanning the CCA region and created before CCA addition; and transcription associated tRFs, formed due to problems in transcription termination
To describe tRFs general populations in wild type ovaries from young flies, we first performed a cascade of annotations of small RNA populations, to the exclusion of rRNA fragments which were previously depleted from the sequence datasets (Figure 2A and Supplementary Figure 8)

Summary

Introduction

Transfer RNAs (tRNAs) are molecules of ∼75 nt transcribed by RNA polymerase III that adopt a typical cloverleaf secondary structure. They are ancient molecules required for protein translation and are encoded by hundreds of genes (∼300 in Drosophila, ∼400 in humans) localized in clusters throughout the genome in some species (Haeusler and Engelke, 2006; Willis and Moir, 2018). RNase P is formed by one RNA molecule and several protein subunits such as Rpp, highly tRNA Fragment Biogenesis Analysis conserved throughout evolution (Jarrous, 2017). RNAse P can cleave non-canonical targets such as rRNA, snoRNA, some long non-coding RNA and RNAs containing N6methyladenosine (m6A) (Coughlin et al, 2008; Jarrous, 2017; Park et al, 2019)

Methods

Results

Conclusion