Temperature-Dependent Regulation of 5-Methylcytosine RNA Modification and Alternative Splicing in Arabidopsis 

J.Q. MA and H. YU 

Department of Biological Sciences and Temasek Life Sciences Laboratory, National University of Singapore 

RNA modification provides a new layer of post-transcriptional regulation of gene expression in living organisms. So far the molecular basis and function of 5-methylcytosine (m5C) RNA modification are largely unknown in plants. Here, we show that an m5C methyltransferase MTC is a “writer” for installing m5C modification in Arabidopsis. Its loss-of-function mutants display developmental defects with a significant decrease of m5C levels under cold stress. We show that MTC interacts with splicing factors including SR45 in a cold stress-dependent manner. Transcriptome analyses revealed that MTC affects hundreds of alternative splicing events under cold stress, including CIRCADIAN CLOCK-ASSOCIATED1 (CCA1) that has been reported to function in cold response via its different splicing variants. We performed qRT-PCR to verify that MTC positively regulates one of the splicing variants of CCA1 in a cold stress-dependent manner. Furthermore, we have performed nanopore sequencing and found that MTC mediates m5C RNA modification on CCA1 under cold stress. We hypothesize that cold stress triggers dynamic m5C modification and alternative splicing medicated by MTC, thus affecting the function of CCA1 and its subsequent effects on cold response. Further investigation will unravel temperature-dependent regulation of m5C RNA modification and the resulting alternative splicing as well as phenotypic alteration.