Elevating RNA m⁵C methylation provides a promising strategy for crop productivity

RNA 5-methylcytidine (m⁵C) has been identified as a key epitranscriptomic modification of mRNAs involved in regulating multiple post-transcriptional processes. Here, we found that knockout of the RNA m⁵C demethylase, OsNOP2, results in elevated m⁵C levels and positively influences numerous agronomic traits in rice. After verifying OsNOP2 RNA m⁵C demethylase function in vitro and in planta, we found that enhanced m⁵C levels arising from OsNOP2 knockout results in increased translation, particularly for transcripts involved in carbon assimilation and nitrogen metabolism. OsNOP2-KO boosts grain yield by ∼28% per plot in the Nipponbare genetic background in normal conditions. Furthermore, these increased yield traits are maintained under both heat treatment and saline soil conditions. More importantly, knockout of OsNOP2 in the rice varieties Longgeng31 and Xiushui134, as well as its orthologs in wheat and tomato, also increases the RNA m⁵C level to enhance yield, supporting functional conservation of OsNOP2's regulatory impacts. Together, our findings unveil an RNA m⁵C-elevating mechanism by OsNOP2 that epigenetically governs carbon assimilation and nitrogen utilization efficiency in plants, providing a potential strategy for genetic improvement in multiple crops.