The Promise of Environmental RNA Research Beyond mRNA.

Environmental RNA (eRNA) studies have primarily focused on species detection and community composition through metabarcoding or metatranscriptomics, and on gene expression through messenger RNA (mRNA) abundance analysis. While valuable, this focus overlooks the broader functional roles of other RNA types in cellular metabolism. Beyond mRNA, noncoding RNAs as well as structural RNAs play critical roles in gene regulation during stress responses, development and adaptation. Additionally, RNA processes like RNA methylation or alternative splicing also respond to similar environmental or developmental signals. When applied to eRNA research, these additional RNA types and RNA processes hold significant potential as powerful, noninvasive tools for monitoring the physiological state of entire species communities. In this roadmap, we present underexplored RNA types and processes relevant for eRNA research, outlining their functions and the challenges of integrating them into the field. Expanding eRNA research to include more diverse aspects of RNA biology will require improved experimental techniques for sensitive and reliable detection and quantification of specific RNAs in eRNA samples, along with enhanced tools for taxonomic and functional annotation and the expansion of genetic reference databases. Where species-level resolution is not possible, functional inferences could be drawn at higher taxonomic levels. Expanding the scope of eRNA studies to encompass more diverse RNA types and RNA processes will provide additional insights into species communities' state, their adaptation potential and responses to stressors in a noninvasive way.