This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
Ribotoxins kill cells by endonucleotically cleaving essential RNAs involved in protein translation (rRNAs, tRNAs, and mRNAs). We have identified a novel bacterial RNA repair system that neutralizes the effect of ribotoxins. This unique RNA repair system appears to not only repair the damage caused by ribotoxins, but also methylates the RNA at the cleavage site so that it cannot be cut again by the ribotoxin. In this project, the bacterial Hen1, a member of the protein complex that performs the repair and methylation, will be the focus of study. In eukaryotes, Hen1 is responsible for 2'-O-methylation on the 3'-terminal nucleotide of some small non-coding RNAs involved in RNA interference. The bacterial Hen1 was found to be one of two proteins that are highly conserved in a sub-set of bacteria. Conserved along with Hen1 is bacterial Pnkp, which has been shown to have kinase, phosphatase, and adenylyltransferase activities that are a hallmark of RNA repair. However, bacterial Pnkp alone was not able to repair damaged RNA. Therefore, the PI hypothesizes that bacteria possessing Hen1 and Pnkp have evolved a clever strategy using the Hen1/Pnkp complex to neutralize the lethal effect of ribotoxins by both repairing the damaged RNA and methylating the RNA to make it resistant to re-cleavage by the ribotoxin. The research in this project consists of two specific objectives: First, the roles of bacterial Hen1 in RNA repair carried out by bacterial Hen1/Pnkp complex will be defined. Both bacterial Hen1 and Pnkp were shown to be required to repair a cleaved tRNA, based on preliminary studies. Therefore, in addition to being part of the ligase activity required for RNA repair, the bacterial Hen1 is proposed here to also play another important role by catalyzing incorporation of a methyl group at the site of damaged RNA during repair. Second, structures of bacterial Hen1 will be determined to help elucidate how Hen1 carries out 2'-O-methylation at the 3'-end of small RNAs. With the determination of crystal structures of the methyltransferase domain of Hen1 alone, mutational studies will be carried out to help analyze Hen 1 function. Furthermore, the PI plans to obtain a crystal structure of Hen1-RNA complex.
The broader impacts of this research include providing insight into biology and biological chemistry in the area of ribotoxins and RNA repair, an emerging field of RNA research. The research will also provide the molecular and mechanistic basis for understanding a new type of RNA methylation by Hen1. In addition, the studies will further our understanding of the role of Hen1 in the function of eukaryotic RNAi due to high homology of the eukaryotic Hen1 to the bacterial Hen1 under study here. The project provides long-term research and education opportunities for undergraduate students, graduate students, and postdoctoral fellows at the interfaces of chemical biology, biochemistry, and structural biology.
