5-Methylcytosine RNA Methylation in &ITArabidopsis Thaliana&IT

5-Methylcytosine (m(5)C) is a well-characterized DNA modification, and is also predominantly reported in abundant non-coding RNAs in both prokaryotes and eukaryotes. However, the distribution and biological functions of m(5)C in plant mRNAs remain largely unknown. Here, we report transcriptome-wide profiling of RNA m(5)C in Arabidopsis thaliana by applying m(5)C RNA immunoprecipitation followed by a deep-sequencing approach (m(5)C-RIP-seq). LC-MS/MS and dot blot analyses reveal a dynamic pattern of m(5)C mRNA modification in various tissues and at different developmental stages. m(5)C-RIP-seq analysis identified 6045 m(5)C peaks in 4465 expressed genes in young seedlings. We found that m(5)C is enriched in coding sequences with two peaks located immediately after start codons and before stop codons, and is associated with mRNAs with low translation activity. We further demonstrated that an RNA (cytosine-5)-methyl-transferase, tRNA-specific methyltransferase 4B (TRM4B), exhibits m(5)C RNA methyltransferase activity. Mutations in TRM4B display defects in root development and decreased m(5)C peaks. TRM4B affects the transcript levels of the genes involved in root development, which is positively correlated with their mRNA stability and m(5)C levels. Our results suggest that m(5)C in mRNA is a new epitranscriptome marker in Arabidopsis, and that regulation of this modification is an integral part of gene regulatory networks underlying plant development.