TB is caused by Mycobacterium tuberculosis (Mtb) and is one of the leading causes of death worldwide. During infection, Mtb can survive and adapt within the host, and this is dependent on the tight regulation of gene expression. mRNA degradation is a major process in this regulation, and it is well characterized in E. coli and B. subtilis, but not in mycobacteria. Therefore, it is fundamental to have a better understanding of mRNA degradation in mycobacteria. We used Mycolicibacterium smegmatis which is non-pathogenic and fast-growing for all our studies. In Chapter 2, we showed a global analysis of the transcriptome organization and post-transcriptional mRNA cleavage landscape in M. smegmatis in log phase and hypoxia conditions. In addition to defining transcription start sites (TSSs), we identified over 3,000 RNA cleavage sites. Importantly, a novel sequence motif was found among these cleavage sites which is different from what has been reported in E. coli. Then we aimed to determine which ribonuclease was the major contributor to these cleavage events, and we hypothesized that it was RNase E. In chapter 3, we determined mRNA half-lives with high confidence when RNase E was normally expressed or repressed. As expected, we showed a global mRNA stabilization when RNase E was repressed. The degree of stabilization varied among transcripts and several potential causes of this variation were assessed, such as mRNA abundance and the 5’ UTRs of transcripts. Importantly, we also mapped the RNase E cleavage sites in vivo and in vitro and found that RNase E cleaved at the sequence RN↓CNU, consistent with the sequence motif identified in chapter 2. These findings demonstrate an important role for RNase E in mRNA degradation. RNase E interacts with proteins to form RNA degradosome in E. coli and the interactions commonly happen in the scaffold domain of RNase E. To better understand mycobacterial RNase E, in chapter 4 we mapped the boundaries of the RNase E catalytic domain and scaffold domains, which are different from E. coli RNase E. The first 330 residues of the N-terminal scaffold domain had roles in the sub-cellular localization of RNase J, cell size, gene expression, and mRNA stabilization. Overall, we have compiled evidence showing that RNase E is important in mRNA degradation in M. smegmatis.

Creator

• Zhou, Ying

Contributors

• Shell, Scarlet

Degree

• PhD

Unit

• Biology & Biotechnology

Publisher

• Worcester Polytechnic Institute

Identifier

• etd-47036

Keyword

• mRNA cleavage
• RNA degradosome
• mRNA degradation
• Mycolicibacterium smegmatis
• Transcriptome
• RNase E

Advisor

• Shell, Scarlet

Orcid

• https://orcid.org/0000-0002-8988-2243

Defense date

• 2022-01-18

Year

• 2022

Date created

• 2022-01-25

Resource type

• Dissertation

Rights statement

• In Copyright