Transcription and RNA turnover shape the pool of transcripts in the cell. Regulation of RNA degradation, however, remains elusive when compared to the enumerable mechanisms of transcriptional control. In bacteria, mRNA decay is thought to be initiated by a few endoribonucleases that have promiscuous activities, yet mRNA half-lives vary greatly. Here I propose to globally define the set of adaptors for endoribonucleases that instruct mRNA decay, akin to transcription factors modulating the activity of RNA polymerase. Only a few adaptors of degradation have been discovered in bacteria, and they were all found serendipitously. To provide a far more comprehensive view, I will develop several general methodologies that will be translatable to other organisms. My first approach will identify classes of RNAs potentially targeted by the same adaptor, using their dependence on nuclease levels as a classifier. Demonstration of this top-down method utilizes the central endonuclease RNase Y from the gram-positive bacterium Bacillus subtilis. Completion of the first part of this aim will be the first high-resolution study of RNA degradation in B. subtilis and demonstrates a general strategy to define the direct targets of promiscuous central endonucleases. Secondly, to complement the systems-wide approach, I have designed both genetic and biochemical approaches to identify the molecular constituents of adaptors of degradation. Discoveries of novel adaptors for mRNA decay will expand our understanding of gene regulation and provide transformative tools for molecular biology. Together, this set of top-down and bottom-up approaches will transform our understanding of endonuclease specificity in bacteria and provide a general strategy for studying the post-transcriptional control of gene expression. The fellowship training plan leverages complementary expertise and experiences of two mentors, Dr. Gene- Wei Li and Dr. Alan Grossman. Technical training within the research strategy consists of (1) quantitative modeling and next-generation sequencing techniques from my sponsor Dr. Li and (2) methodologies in forward genetics assays and nucleic acid biochemistry from my co-sponsor Dr. Grossman. Dr. Li is a trained physicist and has a record of developing cutting-edge approaches to address his unique perspective on gene regulation. Dr. Grossman is a renowned bacterial geneticist and has thirty years of mentorship experience as a professor with many postdoctoral trainees obtaining independent research positions. The combined environment fostered by these two individuals at the Massachusetts Institute of Technology serves to address the aims of the research proposed and to aid in my development as an independent researcher.
Regulated turnover of messenger RNAs is an important mechanism for bacterial pathogens to invade their hosts. By developing techniques to understand the mechanistic basis of messenger RNA turnover in the gram- positive bacterial model organism Bacillus subtilis, this research will provide foundational knowledge on how closely related organisms, such as Staphylococcus aureus and Streptococcus pyogenes, regulate their transcriptome and virulence.
