Widespread occurrence of N6-methyladenosine in bacterial mRNA - PubMed (original) (raw)

. 2015 Jul 27;43(13):6557-67.

doi: 10.1093/nar/gkv596. Epub 2015 Jun 11.

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Widespread occurrence of N6-methyladenosine in bacterial mRNA

Xin Deng et al. Nucleic Acids Res. 2015.

Abstract

N(6)-methyladenosine (m(6)A) is the most abundant internal modification in eukaryotic messenger RNA (mRNA). Recent discoveries of demethylases and specific binding proteins of m(6)A as well as m(6)A methylomes obtained in mammals, yeast and plants have revealed regulatory functions of this RNA modification. Although m(6)A is present in the ribosomal RNA of bacteria, its occurrence in mRNA still remains elusive. Here, we have employed ultra-high pressure liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (UHPLC-QQQ-MS/MS) to calculate the m(6)A/A ratio in mRNA from a wide range of bacterial species, which demonstrates that m(6)A is an abundant mRNA modification in tested bacteria. Subsequent transcriptome-wide m(6)A profiling in Escherichia coli and Pseudomonas aeruginosa revealed a conserved m(6)A pattern that is distinct from those in eukaryotes. Most m(6)A peaks are located inside open reading frames and carry a unique consensus motif of GCCAU. Functional enrichment analysis of bacterial m(6)A peaks indicates that the majority of m(6)A-modified genes are associated with respiration, amino acids metabolism, stress response and small RNAs, suggesting potential functional roles of m(6)A in these pathways.

© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

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Figures

Figure 1.

Figure 1.

Presence of m6A in bacterial mRNA. (A) The m6A/A ratios of mRNA isolated from seven bacterial species. (B) The m6A/A ratios of mRNA isolated from different strains of Escherichia coli and Pseudomonas aeruginosa. Error bars represent standard deviations, which were calculated from three replicates.

Figure 2.

Figure 2.

Overview of m6A methylome in Escherichia coli. (A) The m6A peak distribution within different gene contexts. The y-axis represents (number of reads/length unit)/(number of total reads), which is an indicator of the extent to which sequencing reads are enriched in different segments across the entire transcript. (B) Accumulation of m6A reads along transcripts. Each transcript is divided into three parts: Overlap Start, Inside and Overlap End. (C) The UGCCAG conserved sequence motif for m6A-containing peak regions. (D) Pie chart displaying the percentage of genes containing m6A peaks with functional categories. (E) GO-enrichment analysis of all the genes with m6A peaks. The effect size (number of enriched genes/total genes in the GO category) for each category is 15/45 (aerobic respiration), 15/122 (amino acids biosynthesis), 6/138 (response to abotic stimulus), 5/61 (cell wall biosynthesis) and 6/39 (anaerobic respiration), respectively. The statistical test (_P_-value) used by DAVID was the Fisher Exact test.

Figure 3.

Figure 3.

Accumulation of m6A reads in hyaABCD genes (A), gabDT (B) and lacZI(C) in Escherichia coli transcriptome. CK represents the control sample and IP represents ChIP-seq sample.

Figure 4.

Figure 4.

Overview of m6A methylome in Pseudomonas aeruginosa. (A) The m6A peak distribution within different gene contexts. The y-axis represents (number of reads/length unit)/(number of total reads), which is an indicator of the extent to which sequencing reads are enriched in different segments across the entire transcript. (B) Accumulation of m6A reads along transcripts. Each transcript is divided into three parts: Overlap Start, Inside and Overlap End. (C) The GGCCAG conserved sequence motif for m6A-containing peak regions. (D) Pie chart displaying the percentage of genes containing m6A peaks with functional categories. (E) KEGG-enrichment analysis of all the genes with m6A peaks. The effect size (number of enriched genes/total genes in the KEGG category) for each category is 7/148 (amino acids metabolism), 4/37 (glycolysis) and 3/56 (TCA cycle), respectively.

Figure 5.

Figure 5.

Accumulation of m6A reads in PA3415–3417 and ldh (A), rsmY (B) and rsmZ (C) in Pseudomonas aeruginosa transcriptome. CK represents control sample and IP represents ChIP-seq sample.

Figure 6.

Figure 6.

Growth temperature significantly affects the m6A/A ratio in Pseudomonas aeruginosa. Error bars are calculated from three replicates.

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