RNA-catalyzed synthesis of post-transcriptional RNA modifications

• Applicant: Professorin Dr. Claudia Höbartner
• Subject Area: Biological and Biomimetic Chemistry
• Term: since 2021
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 463143961

Nearly all classes of coding and non-coding RNA are decorated with post-transcriptional modifications, which play important functional roles in all kingdoms of life. Methylated nucleotides belong to the most evolutionary conserved features of RNA, and are installed by specialized protein enzymes that use S-adenosyl-L-methionine (SAM) as primary methyl donor. This and other nucleotide cofactors are considered “evolutionary leftovers” from an RNA world. Methylated nucleotides have been referred to as “molecular fossils” produced on early Earth that could have influenced the evolutionary path of catalytic RNA. The hypothesis suggests that ribozymes may have installed modifications to enhance catalysis, or catalyzed their removal to facilitate replication and storage of genetic information. It may be possible to reconstruct modern analogues of such primordial ribozymes using in vitro evolution methods in the laboratory. This project focusses on the in vitro evolution of ribozymes that catalyze the site-specific installation of RNA modifications, with a special attention on nucleobase and ribose methylation. This proposal builds on our very recent discovery of the first methyltransferase ribozyme, which catalyzes the synthesis of 1-methyladenosine (m1A) using O6-methylguanine (m6G) as the methyl group donor. This finding suggests that additional RNA-modifying ribozymes can be identified. We will focus on natural nucleobase modifications that are found at conserved positions in tRNAs and rRNAs, including m3C, m6A, i6A and 2'-O-methyl nucleosides. In parallel, we will search for ribozymes that can remove methylated nucleotides by demethylation. Such ribozymes would mimic the activity of repair enzymes or “eraser proteins”. The key selection step in all these evolution experiments requires a faithful separation of methylated and unmodified RNA, such that only active RNA sequences will be selectively amplified. We propose that this can be achieved by modification-sensitive deoxyribozymes, which site-specifically cleave either modified or unmodified RNA, but not both. Our recently reported deoxyribozymes that sense m6A or i6A are key enabling tools for the proposed research. In addition, we are interested to understand the function of the first methyltransferase ribozyme in more detail. We will combine chemical, biochemical, and biophysical studies to analyze the structure of the ribozyme and the architecture of the active site. Atomic mutagenesis of the target nucleotide and the cofactor will provide structure-function relationships and mechanistic insight into RNA-catalyzed methyl transfer reactions. Moreover, rational modification of the cofactor, possibly combined with reselection from partially randomized libraries, will enable RNA-mediated post-synthetic installation of bioorthogonal RNA modifications. From this project, we expect critical new insights into the catalytic abilities of RNA and provide useful tools for RNA research.

• DFG Programme: Research Grants