Next-Generation Sequencing-Based RiboMethSeq  Protocol for Analysis of tRNA 2'-O-Methylation

• Published in: Biomolecules (Basel, Switzerland) Vol. 7; no. 1; p. 13
• Main Authors: Marchand, Virginie, Pichot, Florian, Thüring, Kathrin, Ayadi, Lilia, Freund, Isabel, Dalpke, Alexander, Helm, Mark, Motorin, Yuri
• Format: Journal Article
• Language: English
• Published: Switzerland MDPI AG 09-02-2017; MDPI
• Subjects: 2′‐O‐methylation; Biochemistry, Molecular Biology; Computational Biology - methods; deleted strain; E coli; Enzymes; Escherichia coli - chemistry; Escherichia coli - genetics; Genomics; Growth conditions; High-Throughput Nucleotide Sequencing - methods; high‐throughput sequencing; Life Sciences; Mapping; Methylation; Molecular biology; Optimization; RiboMethSeq; RNA modification; RNA, Bacterial - chemistry; RNA, Fungal - chemistry; RNA, Transfer - chemistry; rRNA; Saccharomyces cerevisiae - chemistry; Saccharomyces cerevisiae - genetics; Sequence Analysis, RNA - methods; Species; Transfer RNA; TRM3; TrmH; tRNA; Yeast; 2′‐O‐methylation; TRM3; tRNA; high‐throughput sequencing; deleted strain; RiboMethSeq; TrmH; deleted strain; 2 -O-methylation; high-throughput sequencing
• ISSN: 2218-273X; 2218-273X
• DOI: 10.3390/biom7010013

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.