Detection of stress-dependent m⁵C rRNA dynamics inEscherichia coliusing m⁵C-Rol-LAMP

Numerous RNA modifications are known in prokaryotes, but their dynamics and function in regulation remain largely unexplored. InEscherichia coli, three methyltransferases catalyze the 5-methylcytosine (m⁵C) modification in ribosomal RNA. Here, we introduce m⁵C-rolling circle loop-mediated isothermal amplification (m⁵C-Rol-LAMP) as a novel qPCR-based method that offers high sensitivity and site- specific resolution to detect and quantify m⁵C in total RNA. When applying m⁵C-Rol-LAMP toE. coliunder heat stress (45 °C), we observe a site-specific increase of m⁵C at position 1407 of 16S rRNA from 77% to 89%, while m⁵C levels at positions 967 (16S) and 1962 (23S) remain unchanged. In recovered cells (at 37°C), the m⁵C abundance partially returns to the no stress level. Under oxidative stress, the level of m⁵C1407 also increases, but remains high in recovered cells. These results demonstrate for the first time a reversible, stress-dependent and site-specific change in the rRNA modification level of a bacterium. m⁵C-Rol-LAMP is a powerful and easy-to-use tool for studying m⁵C in all RNA species, allowing the quantitative and site-specific detection of this modification.