The Coordinate Functional Roles of caRNA and mRNA Modification

RNA modifications are critical regulators of gene expression and play indispensable roles in diverse physiological processes. Multiple types of RNA modifications are found on mRNAs, where they interact with a wide range of reader proteins and influence nearly all aspects of mRNA processing. Beyond mRNAs, RNA modifications also occur on specific non-coding RNAs (ncRNAs), adding an additional layer to transcriptional and chromatin regulation. In our previous studies, we discovered that chromatin-associated regulatory RNAs (carRNAs) carry m6A modifications that regulate chromatin states, gene transcriptional activity, and stem cell fate determination. We further identified abundant m6A modifications on centromeric RNAs (cenRNAs) in cancer cells and demonstrated that CENPA maintains centromere integrity during replication by interacting with methylated cenRNAs, revealing an epitranscriptomic mechanism that safeguards centromere stability and provides a potential therapeutic target in cancer. To achieve a finer understanding of the functional diversity and molecular mechanisms of m6A, we developed FOCAS—a CRISPR-dCas13b–based high-throughput, site-specific functional screening platform for m6A. Applying FOCAS to four human cancer cell lines, we identified multiple m6A-regulated genes that influence cell fitness through both mRNAs and ncRNAs. FOCAS further revealed context-dependent and reader-specific effects of m6A within the same gene, uncovering the complex regulatory logic of this modification and establishing a foundation for understanding its mechanistic diversity and therapeutic potential in diseases.