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Access Type
WSU Access
Date of Award
January 2023
Degree Type
Dissertation
Degree Name
Ph.D.
Department
Chemistry
First Advisor
Matthew Allen
Second Advisor
Jared Schrader
Abstract
The bacterial cytoplasm has been assumed to be poorly organized due to a lack of membrane-bound organelles. Biomolecular condensates (hereafter referred to as condensates) were recently discovered as an alternative mechanism to organize the cellular cytoplasm. Condensates are non-membrane-bound organelles that are formed through liquid-liquid phase separation by concentrating a specific set of proteins/nucleic acids to compartmentalize cellular biochemistry. While eukaryotic condensates have been broadly identified and characterized, less is known about the organization and functions of bacterial condensates. Eukaryotic condensates play a critical role in organizing the biochemical steps of gene expression including mRNA transcription, processing, and decay. The first and well studied bacterial condensate being BR-bodies that organize mRNA decay. This dissertation aims to apply a systematic method to define the BR-body proteome and understand the mechanisms by which BR-bodies organize mRNA decay. Using a cellular enrichment by differential centrifugation followed by LC-MS proteomics analysis, we identified >100 proteins enriched in BR-bodies including many proteins known to affect mRNA decay. However, one specific type of protein that is important for mRNA degradation is decapping enzymes (RppH and NudC) that were found to be depleted from BR-bodies. Interestingly, by generating rppH and nudC deletion strains, we found that decapping activity is required for robust BR-body formation. By measuring the global mRNA decay rates utilizing Rif-seq, we observed a significant subset of the C. crescentus transcriptome was turned over more slowly in the rppH deletion strain. GO-term analysis found that RppH regulated mRNAs are enriched in amino acid and carbon metabolism genes. Overall, our results suggest that decapping enzymes induce rapid BR-body formation in cells which could help bacteria to rapidly turnover the transcriptome to shut off genes whose expression is no longer required, thereby adapting to ever changing environmental conditions by regulating translation. Molecular mechanisms of bacterial gene expression remain unclear due to the importance of cellular organization for precise gene expression. However, a comprehensive understanding of BR-body condensates catalyzed mRNA-decay could help in designing new antibiotic targets. Therefore, BR-bodies could be attractive targets for developing next-generation antibiotics to fight antibiotic resistance, a global public health threat.
Recommended Citation
Rathnayaka Mudiyanselage, Prasanjali Imalka Wanigasekara, "The Proteome Of Bacterial Rnp Condensates (br-Bodies) And Role Of 5’ Nucleotide Modifications In Br-Body Assembly And Messenger Rna Decay" (2023). Wayne State University Dissertations. 3935.
https://digitalcommons.wayne.edu/oa_dissertations/3935