Catabolism of non-canonical nucleosides from RNA turnover in plants

• Applicant: Professor Dr. Claus-Peter Witte, since 1/2020
• Subject Area: Plant Biochemistry and Biophysics ; Plant Physiology
• Term: from 2018 to 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 416961553

Over 100 chemical modifications of nucleobases exist in the various types of RNA. In mRNA, the RNA modifications influence for example pre-mRNA splicing, transcript stability, and translation. Upon RNA turnover, the modified nucleosides will be released together with other canonical nucleosides. Cells possess catabolic and recycling pathways to metabolize the canonical nucleosides, however, the metabolic fate of the modified nucleosides is unclear. Recently, we identified an enzyme (MAPDA) which deaminates N6-mAMP derived from mRNA degradation generating inosine monophosphate (IMP). The abrogation of the corresponding gene in plants and in mammalian cells resulted in N6-methyladenosine, N6-mAMP and N6-mATP accumulation in vivo. It is possible that MAPDA and other ‘molecular filters’ prevent the accumulation of aberrant nucleotides to avoid their uncontrolled re-use in nucleic acid biosynthesis. MAPDA is removing N6-mAMP, but what is the metabolic fate of all the other modified nucleosides / nucleotides? This is the principle question to be addressed in this proposal. We will elucidate several potential catabolic pathways for modified nucleosides in Arabidopsis, including those for pseudouridine and 5-methylcytidine. Our preliminary data shows that plants employ two peroxisome-localized enzymes to degrade pseudouridine. The molecular and phenotypic consequences of genetic abrogation of this pathway as well as of its cytosolic mis-localization will be studied in Arabidopsis thaliana. Preliminary data shows that compromising pseudouridine degradation results in a delay of seedling development as well as reduced seed production. For 5-methylcytidine and 5-methyluridine, we have indications that they are degraded via the canonical pyrimidine catabolic pathway. We would like to characterize the catabolism of these two modified nucleosides in detail, and furthermore to elucidate the metabolic fate and catabolism of other modified purines and pyrimidines by analyzing nucleoside / nucleobase profiles in a variety of mutants. Techniques will be established to systematically quantify a broad range of modified nucleosides from Arabidopsis by mass spectrometry. This research will highlight a so far largely overlooked aspect in RNA modification research: Extensive post-transcriptional modification of RNA does not only require the precise placement and maintenance of modifications, but also requires that dedicated catabolic pathways are in place to prevent the accumulation of modified nucleosides avoiding their salvage and possible random re-incorporation into new RNA. In this context the molecular and phenotypic consequences of compromising such catabolic pathways will be investigated.

• DFG Programme: Research Grants
• Ehemaliger Antragsteller: Mingjia Chen, Ph.D., until 1/2020