Biochemical characterization of RNase R 2'-O-methylation sensitivity

RNase R is a member of the RNA exonuclease family that digests RNA in the 3'-5' direction. Previous studies have identified RNase R from Mycoplasma genitalium (MgR) as the only RNA exonuclease that is sensitive to 2'-O-methylation (Nm) modification. However, the mechanism underlying this characteristic is not well understood. In this study, we aimed to explore the molecular mechanism of RNase R Nm sensitivity using an improved assay that can better evaluate Nm sensitivity. By comparing the sequences of five wild-type RNase R variants from Mycoplasma, we identified the importance of loop 18 in Nm sensitivity. Furthermore, we demonstrated the critical roles of L283, T278, and T279 within loop18. Our findings deepen the understanding of the molecular mechanism of why MgR is sensitive to Nm and provide a potential direction of protein engineering for applications.