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

Methylation of carbon‐5 of cytosines (m⁵C) is a post‐transcriptional nucleotide modification of RNA found in all kingdoms of life. While individual m⁵C‐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 m⁵C in RNA, demonstrating that this modification is non‐essential. Using this genetic tool, we determine the localisation and enzymatic specificity of m⁵C 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 m⁵C impacts the decoding of some triplets of these two amino acids, leading to reduced translation efficiency. Upon heat stress, m⁵C loss leads to ribosome stalling at UUG triplets, the only codon translated by an m⁵C34‐modified tRNA. This leads to reduced translation efficiency of UUG‐rich transcripts and impaired fertility, suggesting a role of m⁵C tRNA wobble methylation in the adaptation to higher temperatures.