Next Article in Journal / Special Issue
Cross-Talk between Dnmt2-Dependent tRNA Methylation and Queuosine Modification
Previous Article in Journal / Special Issue
The Escherichia coli COG1738 Member YhhQ Is Involved in 7-Cyanodeazaguanine (preQ0) Transport
Article Menu

Export Article

Open AccessArticle
Biomolecules 2017, 7(1), 13; doi:10.3390/biom7010013

Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation

1
IMoPA UMR7365 CNRS‐UL, Biopole Lorraine University, 54505 Vandoeuvre‐les‐Nancy, France
2
Next‐Generation Sequencing core facility, FR3209 BMCT CNRS‐UL, Biopole Lorraine University, 54505 Vandoeuvre‐les‐Nancy, France
3
Institute of Pharmacy and Biochemistry, Johannes Gutenberg University Mainz, 55128 Mainz, Germany
4
Department of Infectious Diseases, Medical Microbiology and Hygiene, Ruprecht‐Karls University Heidelberg, 69120 Heidelberg, Germany
*
Author to whom correspondence should be addressed.
Received: 29 December 2016 / Accepted: 2 February 2017 / Published: 9 February 2017
(This article belongs to the Collection RNA Modifications)
View Full-Text   |   Download PDF [2429 KB, uploaded 14 February 2017]   |  

Abstract

Analysis of RNA modifications by traditional physico‐chemical approaches is labor intensive, requires substantial amounts of input material and only allows site‐by‐site measurements. The recent development of qualitative and quantitative approaches based on next‐generation sequencing (NGS) opens new perspectives for the analysis of various cellular RNA species. The Illumina sequencing‐based RiboMethSeq protocol was initially developed and successfully applied for mapping of ribosomal RNA (rRNA) 2′‐O‐methylations. This method also gives excellent results in the quantitative analysis of rRNA modifications in different species and under varying growth conditions. However, until now, RiboMethSeq was only employed for rRNA, and the whole sequencing and analysis pipeline was only adapted to this long and rather conserved RNA species. A deep understanding of RNA modification functions requires large and global analysis datasets for other important RNA species, namely for transfer RNAs (tRNAs), which are well known to contain a great variety of functionally‐important modified residues. Here, we evaluated the application of the RiboMethSeq protocol for the analysis of tRNA 2′‐O‐methylation in Escherichia coli and in Saccharomyces cerevisiae. After a careful optimization of the bioinformatic pipeline, RiboMethSeq proved to be suitable for relative quantification of methylation rates for known modified positions in different tRNA species. View Full-Text
Keywords: tRNA; 2′‐O‐methylation; RiboMethSeq; high‐throughput sequencing; deleted strain;  TrmH; TRM3 tRNA; 2′‐O‐methylation; RiboMethSeq; high‐throughput sequencing; deleted strain;  TrmH; TRM3
Figures

Figure 1

This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

Supplementary materials

Scifeed alert for new publications

Never miss any articles matching your research from any publisher
  • Get alerts for new papers matching your research
  • Find out the new papers from selected authors
  • Updated daily for 49'000+ journals and 6000+ publishers
  • Define your Scifeed now

SciFeed Share & Cite This Article

MDPI and ACS Style

Marchand, V.; Pichot, F.; Thüring, K.; Ayadi, L.; Freund, I.; Dalpke, A.; Helm, M.; Motorin, Y. Next‐Generation Sequencing‐Based RiboMethSeq Protocol for Analysis of tRNA 2′‐O‐Methylation. Biomolecules 2017, 7, 13.

Show more citation formats Show less citations formats

Note that from the first issue of 2016, MDPI journals use article numbers instead of page numbers. See further details here.

Related Articles

Article Metrics

Article Access Statistics

1

Comments

[Return to top]
Biomolecules EISSN 2218-273X Published by MDPI AG, Basel, Switzerland RSS E-Mail Table of Contents Alert
Back to Top