Defining the roles of small KH-domain RNA binding proteins in mycobacteria

Worcester Polytechnic Institute received a $562,123 Project Grant from the National Institute of General Medical Sciences (NIGMS) under the Biomedical Research and Research Training program (CFDA 93.859), effective August 1, 2025 through July 31, 2028. This award supports fundamental research to define the roles of small KH-domain RNA binding proteins (KhpA and KhpB) in mycobacteria, specifically using the non-pathogenic model species Mycolicibacterium smegmatis. The research addresses a significant knowledge gap in understanding how mycobacteria regulate gene expression and survive environmental stressors, with findings potentially applicable to pathogenic species such as Mycobacterium tuberculosis.

The project deliverables include determination of the biochemical properties and functional oligomerization states of KhpA and KhpB proteins, identification of messenger RNA (mRNA) and small regulatory RNA (sRNA) binding targets, characterization of RNA sequence properties that make them binding substrates, quantification of the proteins' impact on RNA degradation rates, and assessment of their effects on bacterial growth and stress tolerance. The award also provides in-depth research training opportunities for undergraduate students at Worcester Polytechnic Institute, aligning with NIGMS's mission to support both fundamental biomedical discoveries and the development of the next generation of research scientists.

Abstract

Mycobacteria include important human pathogens such as mycobacterium tuberculosis, and elucidating the mechanisms by which they regulate gene expression is necessary to understand their mechanisms of surviving drugs and other stressors encountered during infection. However, the mycobacteria are evolutionarily divergent from better-studied model organisms. Small RNA binding proteins have critical roles in mediating srna-mrna interactions and modulating mrna stability in e. Coli. However, mycobacteria lack orthologs of the RNA binding proteins that are best characterized in e. Coli. Instead, mycobacteria encode two putative RNA binding proteins, khpa and khpb, that have orthologs in some gram-positive bacteria where their functions are poorly understood. Given the almost complete absence of published data on small RNA binding proteins in mycobacteria, we propose to determine the biochemical properties and functional impact of khpa and khpb using the non-pathogenic model mycobacterial species mycolicibacterium smegmatis. The proposed project will address a major knowledge GAP in the mycobacterial field while providing in-depth training opportunities for undergraduate students at worcester polytechnic institute. We hypothesize that khpa and khpb form both homodimers and heterodimers that bind differing suites of mrnas and small regulatory rnas (srnas), affecting their stability and function. The objectives of this proposal are to determine the functional oligomerization states of these two proteins; determine the compendia of rnas that they bind; identify the properties that make these rnas targets of khpa/b; determine the impact of khpa/b on RNA degradation rates; and determine the impact of khpa/b on growth and stress tolerance.