RNase III in M. smegmatis

Joubran, Samantha R.

Student Work

RNase III in M. smegmatis

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Mycobacterium tuberculosis causes 1.4 million deaths world-wide every year (World Health Organization, 2020). An important aspect of the bacteria’s ability to survive environmental or antibiotic stressors is its regulation of RNA metabolism. Ribonuclease III is an enzyme that cleaves double-stranded RNA. In some bacteria, it plays an important role in mRNA degradation as well as tRNA and rRNA maturation. It is an essential gene in Mycobacterium smegmatis, a non-pathogenic model for Mycobacterium tuberculosis, and this project focused on understanding this essentiality in the context of mRNA degradation and rRNA maturation. Potential RNase III mRNA cleavage sites were determined and the expression levels of the genes flanking these sites were quantified when RNase III was knocked down. The initial studies showed that RNase III had no effect on the expression of the genes flanking these sites. The focus of the project shifted to examining the role of RNase III in rRNA maturation. RNA agarose gels of samples where RNase III was knocked down showed new bands at around 5,000 base pairs appearing and a suspected pre-16S rRNA band disappearing compared to the control samples. 5’ RACE and 3’ RACE experiments confirmed the presence of a cleavage site 151 bp upstream of the 16S rRNA gene (MSMEG_3757) and another cleavage site 212 bp upstream of the 23S rRNA gene (MSMEG_3756); both are hypothesized to be done by RNase III. Additionally, these experiments identified the location of another cleavage site 150 bp upstream of the 23S rRNA gene, which is likely the result of RNase E. Further testing will need to be done in order to confirm that these sites are cleaved by RNase III and RNase E.

This report represents the work of one or more WPI undergraduate students submitted to the faculty as evidence of completion of a degree requirement. WPI routinely publishes these reports on its website without editorial or peer review.

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