Translational adaptation to heat stress is mediated by RNA 5-methylcytosine in Caenorhabditis elegans

Methylation of carbon-5 of cytosines (mC) is a post-transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual mC-methyltransferases have been studied, the impact of the global cytosine-5 methylome on development, homeostasis and stress remains unknown. Here, using Caenorhabditis elegans, we generated the first organism devoid of mC in RNA, demonstrating that this modification is non-essential. Using this genetic tool, we determine the localisation and enzymatic specificity of mC sites in the RNome in vivo. We find that NSUN-4 acts as a dual rRNA and tRNA methyltransferase in C. elegans mitochondria. In agreement with leucine and proline being the most frequently methylated tRNA isoacceptors, loss of mC impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, mC loss leads to ribosome stalling at UUG triplets, the only codon translated by an mC34-modified tRNA. This leads to reduced translation efficiency of UUG-rich transcripts and impaired fertility, suggesting a role of mC tRNA wobble methylation in the adaptation to higher temperatures.