Unmasking the modified small non-coding RNome: detection strategies for RNA modifications

RNA molecules carry a wide range of chemical modifications that play key roles in regulating their structure, stability, and function. These modifications are especially abundant in small and non-coding RNAs, such as transfer RNAs (tRNA), ribosomal RNA (rRNA), and related fragments, where they influence processes from translation to gene silencing. However, studying these modifications remains challenging due to the short length of these RNAs, their strong secondary structures, and the high density of chemical marks, all of which can interfere with standard sequencing workflows. This review summarizes current strategies for detecting and mapping RNA modifications in the small RNome. We outline the diversity of RNA classes and their characteristic modification patterns, and then discuss key methodological advances. These include demodification-based sequencing approaches for heavily modified RNAs, targeted chemical and enzymatic methods for site-specific mapping, and emerging direct RNA sequencing technologies that allow analysis of native molecules without prior conversion. We also highlight orthogonal validation techniques used to confirm modification identity and improve reliability. Together, these approaches provide complementary insights, but no single method is sufficient on its own. Careful experimental design and validation therefore remain essential for accurate and comprehensive analysis of RNA modifications.