Opportunities and Challenges to Profile mRNA Modifications in Escherichia coli**

Abstract

mRNA methylation is an important regulator of many physiological processes in eukaryotes but has not been studied in depth in prokaryotes. Working with bacterial mRNA is challenging because it lacks a poly(A)‐tail. However, methods for detecting RNA modifications, both sequencing and mass spectrometry, rely on efficient preparation of mRNA. Here, we compared size‐dependent separation by electrophoresis and rRNA depletion for enrichment of Escherichia coli mRNA. The purification success was monitored by qRT‐PCR and RNA sequencing. Neither method allowed complete removal of rRNA. Nevertheless, we were able to quantitatively analyze several modified nucleosides in the different RNA types. We found evidence for stress dependent RNA modification reprofiling in rRNA, but also several modified nucleosides in the mRNA enriched fractions showed significant changes.

Dynamics of diversified A-to-I editing inStreptococcus pyogenesis governed by changes in mRNA stability

“…The dynamic nature of RNA modifications has attracted considerable interest as a means of stress-dependent regulation of gene expression in eukaryotes (76). In this study, we identified several infection-relevant stress conditions that caused substantial alterations in the editing levels of S. pyogenes , adding to the growing body of evidence that bacterial mRNA modifications are similarly dynamic (77–79). Although condition-dependent A-to-I editing has previously been reported for bacteria, most work has focused on a single selected target gene and has not provided a more comprehensive view of the dynamics of the editome (21, 23, 24).…”

Depletion of m6A-RNA inEscherichia colireduces the infectious potential of T5 bacteriophage

“…To identify m 6 A methylation of the rRNA and mRNA moieties in wild type and Δ rlmF Δ rlmJ mutant V. campbellii , total RNA was isolated and processed as previously described (12). In brief, V. campbellii cells were grown to an optical density at 600nm (OD 600 ) of 1 and harvested by centrifugation.…”

“…Total RNA was isolated and tRNA was removed by size exclusion chromatography, resulting in a fraction containing mRNA and rRNA moieties. This RNA fraction was hydrolyzed and quantitatively analyzed by liquid chromatography-tandem MS (LC-MS/MS) (12).…”

“…Adenosine methylation at the nitrogen ( N 6 -methyladenosine - m 6 A) is present in all types of Escherichia coli RNA (12), however, the methyltransferases are only well documented for ribosomal RNA (rRNA) and tRNA. In E. coli , 23S rRNA molecules are methylated at nucleotides A1618 and A2030 by RlmF and RlmJ, respectively (13, 14).…”

Decoding theEscherichia coliepitranscriptome

“…These modifications affect the tRNA structure [5], mediate interactions with aminoacyl tRNA synthetases and elongation factor thermo unstable (EF-Tu) [6,7], and influence the affinity of the anticodon region for specific codons [8]. Previous results from our laboratory have demonstrated the presence of numerous modification types in E. coli mRNA, which vary between growth stages and in response to stress conditions [9]. Despite these earlier findings, bacterial mRNA modifications remain to be explored in detail; their roles, abundance, and distribution are still largely unknown.…”