Identification and functional analysis of RNA modifications in Arabidopsis thaliana and Mus musculus

Abstract

The epitranscriptome is the collection of chemical modifications on ribonucleic acid (RNA) and their molecular functions. These modifications are still being elucidated, and one of these RNA modifications is 5-methylcytosine (m⁵C) and has been shown to affect various cellular activities in animals and plants. However, there are many unanswered questions, in terms of the molecular mechanism, selectivity, dynamics and even catalysing methyltransferases. This thesis aimed to identify the m⁵C sites on the transcriptomes in animals and plants. Bisulphite treatment combined with high throughput sequencing, BS-RNA-Seq, was used to detect m⁵C sites transcriptome-wide at single nucleotide resolution in mouse granulosa cells and in Arabidopsis thaliana seedings. m⁵C sites either in mouse or Arabidopsis datasets were mostly on mRNAs and many were located in clusters within 20 nt. Interestingly, the methylation levels of clustered sites were similar to each other. The distribution of m5C sites on mRNAs was different between mouse and Arabidopsis. In mouse granulosa cells, m⁵C was mainly distributed in the 5’ untranslated region (5’UTR) and coding sequencing (CDS) regions, in contrast to Arabidopsis, the sites were enriched in the CDS. In order to further understand the mechanism underlying m⁵C, motif analysis around m⁵C sites revealed CU-rich sequences. Further, analysis of m⁵C sites by BS-RNA-Seq in two Arabidopsis methyltransferase mutants, rcmt9 and trm4a, identified hundreds of Rcmt9- and Trm4a-dependent sites. Interestingly, many of these sites overlapped. Finally, we explored the role of tRNA Na-methylguanosine (m¹G) methyltransferase TRM5 in Arabidopsis by using a combination of genetics, biochemistry, transcriptomics, proteomics and mass spectrometry. AtTRM5 was localized to the nucleus of plant cells and in yeast complemented the Δtrm5 mutant. In Arabidopsis trm5 mutant, reduced levels assays demonstrated that TRM5 is a bifunctional tRNA guanine and iniosine-N1-methyltransferase. In conclusion, this thesis comprised various perspectives on the influence of RNA modifications, from the genetics of RNA modifying enzymes to the effect on biological processes. These studies demonstrated the important role of RNA modifications on plant growth and development, and the dynamics during mouse gastrulation.