Detection of stress-dependent m5C rRNA dynamics in Escherichia coli using m5C-Rol-LAMP.

Numerous RNA modifications are known in prokaryotes, but their dynamics and function in regulation remain largely unexplored. In Escherichia 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 to E. coli under 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.