Circular RNAs orchestrate integrated post-transcriptional responses to combined heat and drought stress in rice.

Circular RNAs (circRNAs) are emerging post-transcriptional regulators, yet their landscape and functional roles in rice under combined abiotic stress remain largely unexplored. Here, we systematically reanalyzed strand-specific RNA-seq data to characterize circRNAs responsive to simultaneous heat and drought stress. Following quality control, read mapping, and dual-algorithm prediction using CIRI2 and CIRCexplorer2, we identified 208 high-confidence circRNAs distributed across all 12 chromosomes. Comparative profiling revealed 83 circRNAs uniquely expressed in control samples, 51 in stressed samples, and 74 shared between conditions, indicating stress-dependent circularization. Junction-read analysis highlighted a spectrum of circularization strength, ranging from highly abundant circRNAs with dominant junction reads to low-confidence candidates masked by linear transcript background. Genomic annotation showed that circRNAs primarily originated from exonic and intergenic regions, with a pronounced negative-strand bias; several genes generated multiple circRNA isoforms via alternative back-splicing. Functional enrichment of host genes suggested involvement in protein folding, nutrient reservoir activity, RNA degradation, and branched-chain amino acid catabolism, implicating roles in stress adaptation and metabolic regulation. Differential expression analysis identified seven circRNAs specifically induced under combined stress conditions. Network topology analysis pinpointed key miRNAs-including osa-miR414, osa-miR1439, and osa-miR2919-as candidate topological hubs within the predicted network. Their predicted target genes, such as those encoding stress-responsive transcription factors and signaling proteins, suggest potential roles in coordinating post-transcriptional responses to combined stress. Network topology analysis pinpointed key miRNAs-including osa-miR414, osa-miR1439, and osa-miR2919-as candidate topological hubs within the predicted network. Their predicted target genes, such as those encoding stress-responsive transcription factors and signaling proteins, suggest potential roles in coordinating post-transcriptional responses to combined stress. Overall, this study provides a comprehensive map of circRNAs in rice under combined heat and drought stress, suggests their potential as ceRNAs based on predictive analysis, and lays a foundation for future experimental validation of circRNA-mediated regulation.