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Nucleic Acids Res. 2019 Jun 20;47(11):5892-5905. doi: 10.1093/nar/gkz251.

Proteomic and transcriptomic experiments reveal an essential role of RNA degradosome complexes in shaping the transcriptome of Mycobacterium tuberculosis.

Author information

1
Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Pawińskiego 5A, Warsaw 02-106, Poland.
2
Mill Hill Laboratory, Francis Crick Institute, The Ridgeway, Mill Hill, London NW7 1AA, UK.
3
Institute of Medical Biology, Polish Academy of Sciences, Lodowa 106, Łódź 93-232, Poland.
4
Biobank Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, Pilarskiego 14/16, Łódź 90-231, Poland.
5
Institute of Genetics and Biotechnology, University of Warsaw, Pawińskiego 5A, Warsaw 02-106, Poland.

Abstract

The phenotypic adjustments of Mycobacterium tuberculosis are commonly inferred from the analysis of transcript abundance. While mechanisms of transcriptional regulation have been extensively analysed in mycobacteria, little is known about mechanisms that shape the transcriptome by regulating RNA decay rates. The aim of the present study is to identify the core components of the RNA degradosome of M. tuberculosis and to analyse their function in RNA metabolism. Using an approach involving cross-linking to 4-thiouridine-labelled RNA, we mapped the mycobacterial RNA-bound proteome and identified degradosome-related enzymes polynucleotide phosphorylase (PNPase), ATP-dependent RNA helicase (RhlE), ribonuclease E (RNase E) and ribonuclease J (RNase J) as major components. We then carried out affinity purification of eGFP-tagged recombinant constructs to identify protein-protein interactions. This identified further interactions with cold-shock proteins and novel KH-domain proteins. Engineering and transcriptional profiling of strains with a reduced level of expression of core degradosome ribonucleases provided evidence of important pleiotropic roles of the enzymes in mycobacterial RNA metabolism highlighting their potential vulnerability as drug targets.

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