Search results for "Transfer RNA"

Statistically robust methylation calling for whole-transcriptome bisulfite sequencing reveals distinct methylation patterns for mouse RNAs

2017

AbstractCytosine-5 RNA methylation plays an important role in several biologically and pathologically relevant processes. However, owing to methodological limitations, the transcriptome-wide distribution of this mark has remained largely unknown. We previously established RNA bisulfite sequencing as a method for the analysis of RNA cytosine-5 methylation patterns at single-base resolution. More recently, next-generation sequencing has provided opportunities to establish transcriptome-wide maps of this modification. Here we present a computational approach that integrates tailored filtering and data-driven statistical modeling to eliminate many of the artifacts that are known to be associate…

Editorial: RNA modifications – what to read first?

2017

This special issue is dedicated to my favourite pioneer in the world of nucleic acid modifications. Thank you, Henri Grosjean!A stupendous boost in the field of nucleic acid modification has recent...

2016

Urm1 is a unique dual-function member of the ubiquitin protein family and conserved from yeast to man. It acts both as a protein modifier in ubiquitin-like urmylation and as a sulfur donor for tRNA thiolation, which in concert with the Elongator pathway forms 5-methoxy-carbonyl-methyl-2-thio (mcm5s2) modified wobble uridines (U34) in anticodons. Using Saccharomyces cerevisiae as a model to study a relationship between these two functions, we examined whether cultivation temperature and sulfur supply previously implicated in the tRNA thiolation branch of the URM1 pathway also contribute to proper urmylation. Monitoring Urm1 conjugation, we found urmylation of the peroxiredoxin Ahp1 is suppre…

Draft genome sequence of Shimia marina CECT 7688T

2016

Shimia marina is a member of the family Rhodobacteraceae described in 2006. Strain CL-TA03(T) (=CECT 7688(T)) was isolated from a biofilm formed on an acrylic slide submerged in surface water in a coastal fish farm in Tongyeong, Korea. Here we report the draft genome sequence and annotation of S. marina CECT 7688(T) which is composed by 4,001,860bp arranged in 45 scaffolds with a G+C content of 57.4%, 3878 protein coding genes, 40 tRNA genes, 4 rRNA genes and 1 repeat region. An overview of annotated genes revealed diverse genes encoding for exopolysaccharide and capsular biosynthesis enzymes, secondary metabolite biosynthesis enzymes, multiple antibiotic and metal resistance and the abilit…

Differentially Expressed tRNA-Derived Small RNAs Co-Sediment Primarily with Non-Polysomal Fractions in Drosophila

2017

Recent studies point to the existence of poorly characterized small regulatory RNAs generated from mRNAs, rRNAs and tRNAs. To explore the subcellular location of tRNA-derived small RNAs, 0–1 and 7–8 h Drosophila embryos were fractionated on sucrose density gradients. Analysis of 12,553,921 deep-sequencing reads from unfractionated and fractionated Drosophila embryos has revealed that tRFs, which are detected mainly from the 5’ends of tRNAs, co-sediment with the non-polysomal fractions. Interestingly, the expression levels of a subset of tRFs change temporally following thematernal-to-zygotic transition in embryos. We detected non-polysomal association of tRFs in S2 cells as well. Differenti…

Holistic Optimization of Bioinformatic Analysis Pipeline for Detection and Quantification of 2′-O-Methylations in RNA by RiboMethSeq

2020

International audience; A major trend in the epitranscriptomics field over the last 5 years has been the high-throughput analysis of RNA modifications by a combination of specific chemical treatment(s), followed by library preparation and deep sequencing. Multiple protocols have been described for several important RNA modifications, such as 5-methylcytosine (m5C), pseudouridine (ψ), 1-methyladenosine (m1A), and 2'-O-methylation (Nm). One commonly used method is the alkaline cleavage-based RiboMethSeq protocol, where positions of reads' 5'-ends are used to distinguish nucleotides protected by ribose methylation. This method was successfully applied to detect and quantify Nm residues in vari…

MultiBacTAG-Genetic Code Expansion Using the Baculovirus Expression System in Sf21 Cells

2018

The combination of genetic code expansion (GCE) and baculovirus-based protein expression in Spodoptera frugiperda cells is a powerful tool to express multiprotein complexes with site-specifically introduced noncanonical amino acids. This protocol describes the integration of synthetase and tRNA gene indispensable for GCE into the backbone of the Bacmid, the Tn7-mediated transposition of various genes of interest, as well as the final expression of protein using the MultiBacTAG system with different noncanonical amino acids.

RNA Modifications Modulate Activation of Innate Toll-Like Receptors

2019

Self/foreign discrimination by the innate immune system depends on receptors that identify molecular patterns as associated to pathogens. Among others, this group includes endosomal Toll-like receptors, among which Toll-like receptors (TLR) 3, 7, 8, and 13 recognize and discriminate mammalian from microbial, potentially pathogen-associated, RNA. One of the discriminatory principles is the recognition of endogenous RNA modifications. Previous work has identified a couple of RNA modifications that impede activation of TLR signaling when incorporated in synthetic RNA molecules. Of note, work that is more recent has now shown that RNA modifications in their naturally occurring context can have …

Non-Redundant tRNA Reference Sequences for Deep Sequencing Analysis of tRNA Abundance and Epitranscriptomic RNA Modifications

2021

Analysis of RNA by deep-sequencing approaches has found widespread application in modern biology. In addition to measurements of RNA abundance under various physiological conditions, such techniques are now widely used for mapping and quantification of RNA modifications. Transfer RNA (tRNA) molecules are among the frequent targets of such investigation, since they contain multiple modified residues. However, the major challenge in tRNA examination is related to a large number of duplicated and point-mutated genes encoding those RNA molecules. Moreover, the existence of multiple isoacceptors/isodecoders complicates both the analysis and read mapping. Existing databases for tRNA sequencing pr…

2'-O-methylation within prokaryotic and eukaryotic tRNA inhibits innate immune activation by endosomal Toll-like receptors but does not affect recogn…

2019

Bacterial RNA has emerged as an important activator of innate immune responses by stimulating Toll-like receptors TLR7 and TLR8 in humans. Guanosine 2′-O-methylation at position 18 (Gm18) in bacterial tRNA was shown to antagonize tRNA-induced TLR7/8 activation, suggesting a potential role of Gm18 as an immune escape mechanism. This modification also occurs in eukaryotic tRNA, yet a physiological immune function remained to be tested. We therefore set out to investigate the immune modulatory role of Gm18 in both prokaryotic and eukaryotic microorganisms, Escherichia coli and Saccharomyces cerevisiae, and in human cells. Using RiboMethSeq analysis we show that mutation of trmH in E. coli, trm…