Transcriptome maps of general eukaryotic RNA degradation factors and identification and functional characterization of the novel mRNA modification N³-methylcytidine

The first part of this dissertation focused on the different RNA degradation pathways in the yeast Saccharomyces cerevisiae (S. cerevisiae). We used the method photoactivatable ribonucleoside-enhanced crosslinking and immunoprecipitation (PAR-CLIP) to systematically generate transcriptomewide protein binding profiles for 30 general RNA degradation factors. In-depth bioinformatic analysis and comparison with previously reported PAR-CLIP data provided factor enrichment on different RNA classes and the binding behavior for mRNAs and their associated antisense transcripts. The results also gave insights into how the various degradation complexes, and different subunits in these complexes, may be involved in the processing and degradation of different RNA species. Several conclusions were drawn with respect to degradation pathway selection, new functions for known factors were proposed, and several hypotheses have emerged that may be tested in the future. The generated datasets provide a rich resource for future studies of eukaryotic RNA degradation pathways, mechanisms, and the integration of mRNA metabolism. The second part of this dissertation addressed the finding of a novel mRNA modification within the emerging field of epitranscriptomics. We performed PAR-CLIP experiments of the N3-methylcytidine (m3C) tRNA methyltransferase Trm140 to investigate its mRNA binding in the yeast S. cerevisiae. Using this approach in combination with a newly developed method applying anti-m3C antibodies for RNA immunoprecipitation (m3C-CLIP), we were able to call novel m3C modification sites. Next generation sequencing methods and biochemical verification assays showed the m3C modification in tRNA Arginine with anticodon CCU in addition to the established modification sites in tRNA Serine and Threonine in yeast. Our genome wide datasets also provided the first evidence that the m3C modification is present in yeast mRNAs and we mapped the modification on nucleotide resolution. Functional analyses suggested an effect of m3C mRNA modification on RNA degradation mediated by translation efficiency changes. Mapping of the m3C modification sites in human RNA revealed a similar localization compared to yeast over the entire mRNA with higher occupancy towards the end of protein coding transcripts and within CG rich sequences.2020-05-0