Imaging rRNA Methylation in Bacteria by MR-FISH

Methods Mol Biol. 2019:2038:89-107. doi: 10.1007/978-1-4939-9674-2_7.

Abstract

Methylation of RNA is normally monitored in purified cell lysates using next-generation sequencing, gel electrophoresis, or mass spectrometry as readouts. These bulk methods require the RNA from ~104 to 107 cells to be pooled to generate sufficient material for analysis. Here we describe a method-methylation-sensitive RNA in situ hybridization (MR-FISH)-that assays rRNA methylation in bacteria on a cell-by-cell basis, using methylation-sensitive hybridization probes and fluorescence microscopy. We outline step-by-step protocols for designing probes, in situ hybridization, and analysis of data using freely available code.

Keywords: Antibiotic resistance; Automated image analysis; Bacteriology; DNA probes; Epitranscriptomic modifications; FISH; Fluorescence imaging; In situ hybridization; Microscopy; RNA methylation; RNA modification; Ribosomal RNA; Single-cell methods.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Escherichia coli / genetics
  • Escherichia coli / metabolism*
  • Gene Expression Regulation, Bacterial
  • In Situ Hybridization, Fluorescence*
  • Methylation
  • Microscopy, Fluorescence*
  • Molecular Imaging / methods*
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism*
  • RNA, Ribosomal / genetics
  • RNA, Ribosomal / metabolism*
  • Single-Cell Analysis / methods*
  • Time Factors

Substances

  • RNA, Bacterial
  • RNA, Ribosomal