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Regulatory RNAs and their role in Bacillus subtilis

Principal Supervisor: Emma Denham - WMS

Co-supervisor: Andrew Millard and Chrystala Constantinidou

PhD project title: Regulatory RNAs and their role in Bacillus subtilis

University of Registration: University of Warwick

Project outline:

Regulatory RNAs have been identified in all species where they have been looked for and they are bringing the central dogma of molecular biology in to question. The Gram-positive model organism Bacillus subtilis transcribes approximately 1500 condition dependent RNAs that don’t have protein coding capacity1. These RNA molecules can be divided into different classes and include, untranslated regions at the 5’ and 3’ ends of transcripts, sRNAs and antisense RNAs. Several of these RNAs have been studied in B. subtilis and shown to play important roles in the lifestyles of this bacterium. These include regulation of growth phase2, nutrient availability, survival during stress and iron starvation3,4.

Understanding of the role regulatory RNAs play in the biology of Gram-positive organisms is lagging behind that of their Gram-negative counterparts. B. subtilis is an ideal model in which to study regulatory RNAs due to its genetic manipulability, a large knowledge base for its biology and its relation to important pathogens S. aureus and B. anthracis.

Many questions are to be answered in how this organism manages its RNA regulation, including what cellular factor(s) is involved in the RNA-RNA interactions that result in the post-transcriptional regulation. In many Gram-negative organisms the RNA chaperone Hfq is critical to many of these interactions and has enabled much progress in this field. As so little is known regarding the role of regulatory RNAs in B. subtilis this project is flexible in that it can go about answering any of the following questions:

  • What is the role of non-coding RNAs in the lifestyles of B. subtilis (sporulation, stress etc)? Can we use global analysis of RNA-RNA interactions to answer this question?
  • For sRNAs - what are the target/s for sRNAs regulating? Do the sRNAs have single or multiple targets? How are the sRNAs regulating their target/s? Is this a positive or negative regulation?
  • Are there RNA chaperone/s present in the cell? If so what are they and what role do they play in the RNA balance within the cell? Is it involved in how the regulatory RNA finds its target within the cell?
  • What is the role of antisense RNAs? Can we develop a novel mutagenesis system, for example using CRISPR-CAS to disrupt transcription of antisense RNAs and therefore allow their study in the biology of the cell?
  • What is the role of 5’ and 3’ untranslated regions in regulatory events in the cell. Are some of these RNAs processed, as in Gram-negative bacteria to function as sRNAs?
  • Development of novel techniques to identify targets of regulatory RNAs.
  • Understanding the role of any RNA binding proteins involved in the interaction of regulatory RNAs and their targets.

References:

  1. Nicolas et al., Condition-dependent transcriptome reveals high-level regulatory architecture in Bacillus subtilis. Science 335, 1103 (Mar 2, 2012).
  2. Mars et al., Small regulatory RNA-induced growth rate heterogeneity of Bacillus subtilis PLoS Genetics 2015 19;11(3):e1005046
  3. A. Gaballa et al., The Bacillus subtilis iron-sparing response is mediated by a Fur-regulated small RNA and three small, basic proteins. Proceedings of the National Academy of Sciences of the United States of America 105, 11927 (Aug 19, 2008).
  4. S. Brantl, R. Bruckner, Small regulatory RNAs from low-GC Gram-positive bacteria. RNA biology 11, 443 (May, 2014).

BBSRC Strategic Research Priority: Molecules, cells and systems

Techniques that will be undertaken during the project:

  • Molecular and Microbiology
  • RNA-seq
  • Microscopy
  • Flow Cytometry
  • Fluorescence based assays
  • Proteomics
  • RNA analysis and bioinformatics

Contact: Dr Emma Denham, University of Warwick