Dynamic expression and functional transformation of microRNA isoforms induced by osmotic stress in invasive Ciona robusta

Abstract Understanding the mechanisms underlying invasion success is crucial for addressing the rapidly increasing frequency of biological invasions and their escalating ecological and economic impacts worldwide. Phenotypic plasticity plays a crucial role in facilitating invasion success by enabling organisms to respond rapidly to environmental fluctuations. Among molecular regulators of such plasticity, microRNAs (miRNAs) mediate stress adaptation through post-transcriptional gene regulation. Increasing evidence suggests that a single miRNA locus can generate multiple variants (isomiRs), which may diversify regulatory functions and enhance environmental resilience. However, their expression dynamics and functional significance under environmental stress remain largely unexplored during biological invasions. Here, using the highly invasive ascidian Ciona robusta as a model, we examined the dynamic expression and functional divergence of miRNA isoforms in response to osmotic stress. Integrative analysis of miRNAome and transcriptome revealed 10 miRNAs that produced 5’ isomiRs with time-dependent and stress-specific expression patterns. Both canonical miRNAs and their isomiRs targeted overlapping yet distinct gene sets, particularly in pathways related to free amino acid metabolism and ion transport. Functional analysis demonstrated that isomiRs underwent neo-functionalization, sub-functionalization, or mixed functional shifts relative to their canonical counterparts, and in some cases exerted opposite regulatory effects on the same target genes. These results reveal that osmotic stress induces rapid diversification and functional transformation of miRNA isoforms, forming a flexible and dynamic regulatory network. Such plasticity in isomiR regulation likely contributes to enhanced stress tolerance and environmental adaptability, thereby promoting invasion success across diverse, harsh, or rapidly changing environments.