MeRIP-seq detects temperature related variation in methylated environmental RNA in fish

Environmental RNA (eRNA) enables non-invasive, near-real-time and potentially functional insight into aquatic communities by capturing RNA molecules released by living organisms. While most eRNA studies focus solely on transcript abundance, this overlooks other regulatory layers that shape how organisms respond to stress. RNA modifications, particularly N ⁶ -methyladenosine (m ⁶ A), influence RNA stability, processing, and translation and are known to change under temperature challenges. These properties make m ⁶ A a compelling target for eRNA-based biomonitoring. We investigated whether m ⁶ A marks can be recovered from waterborne eRNA released by an African cichlid fish during elevated temperature exposure. eRNA was collected from tank water under control and thermal treatments, enriched for methylated RNA fragments by immunoprecipitation, and sequenced using immunoprecipitation-based MeRIP-Sequencing. Using this approach, we achieved remarkably high read mapping rates to the target fish species, compared to previous studies focusing solely on mRNA sequencing. Moreover, we detect 412 eRNAs having differing abundances between the elevated temperature and control group. Gene ontology analysis reveals that these are enriched in functional categories related to thermal physiology, suggesting a role in the organism’s response to temperature stress. These results indicate an enormous potential of this approach to effectively and non-invasively capture signals of temperature stress response of fish via eRNA. While the current modest replicate depth constrained robust assessment of heat-associated changes in m ⁶ A, to our knowledge, this is the first attempt to assess m ⁶ A RNA modification in the context of eRNA research and the first evidence indicating that extra-organismal eRNA can retain epitranscriptomic information.