Competition between organisms, microbes in particular, for limited resources of nutrition is a fundamental biological phenomenon. To ensure a significant share of nutrition, organisms employ a variety of toxins to eliminate the competition. The targeted organisms in turn employ several defense mechanisms to stay alive. Among various toxins, ribotoxins constitute one of the biggest groups due to their effectiveness in disabling the essential and universally conserved protein translation system. And RNA repair appears to be the defense mechanism employed by certain organisms as antidotes of ribotoxins. However, our understanding of both RNA damage inflicted by ribotoxins and RNA repair by cellular repair systems is limited. Therefore, the overall goals of this application are to identify in vivo RNA targets of a significant number of new ribotoxins as well as several RNA repair systems on a genome-wide scale, and to elucidate molecular mechanisms of RNA damage and repair carried out by new ribotoxins and new RNA repair systems. Employing RNA-seq as our main tool, combined with approaches of bioinformatics, microbiology, biochemistry, and structural biology, we aim to pursue the following lines of investigation: (1) We will perform bioinformatic analyses to identify candidates of new ribotoxins and RNA repair systems; (2) We will employ RNA-seq to pinpoint sites of RNA damage by new ribotoxins; (3) We will employ RNA-seq to interrogate each repair system on its ability to repair diverse damaged RNAs inflicted by various ribotoxins; and (4) We will validate RNA-seq results with in vitro biochemical characterization and carry out structural studies of various components of RNA damage and repair. If toxin-antitoxin (TA) systems are included, it is almost certain that virtually all bacteria species possess ribotoxins. If the proposed RNA repair systems are experimentally verified, we estimate that approximately 40% of bacterial species possess RNA repair capability. Finally, based on our bioinformatic analysis present in this application, RNA repair is likely to occur in eukaryotic organisms.
Like some small-molecule antibiotics, ribotoxins target protein translation system for cell killing. Therefore, those new ribotoxins discovered from our study that selectively inhibit protein translation in pathogens but not in humans might be further developed into therapeutic agents. In addition, six bacteria living in gingival plaque of the human mouth possess a unique RNA repair complex. Thus, blocking RNA repair in these bacteria with small-molecule inhibitors may lead to their demise, resulting in potential prevention and treatment of gum and dental diseases.
