Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides
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Hia-2014-Mycobacterial RNA is.pdf
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Author(s)
Hia, Fabian
Chionh, Yok Hian
Pang, Yan Ling Joy
DeMott, Michael S.
McBee, Megan E.
Dedon, Peter C.
Date Issued
December 2014
Journal
Nucleic Acids Research
Publisher
Oxford University Press
Citation
Hia, F., Y. H. Chionh, Y. L. J. Pang, M. S. DeMott, M. E. McBee, and P. C. Dedon. “Mycobacterial RNA Isolation Optimized for Non-Coding RNA: High Fidelity Isolation of 5S rRNA from Mycobacterium Bovis BCG Reveals Novel Post-Transcriptional Processing and a Complete Spectrum of Modified Ribonucleosides.” Nucleic Acids Research (December 24, 2014).
Version
Final published version
Abstract
A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria.
MIT Department
Massachusetts Institute of Technology. Center for Environmental Health Sciences
Massachusetts Institute of Technology. Department of Biological Engineering
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DOI of Published Version
https://doi.org/10.1093/nar/gku1317
