At-RS31 orchestrates hierarchical cross-regulation of splicing factors and integrates alternative splicing with TOR-ABA pathways.

Alternative splicing is essential for plants, enabling a single gene to produce multiple transcript variants to boost functional diversity and fine-tune responses to environmental and developmental cues. Arabidopsis thaliana At-RS31, a plant-specific splicing factor in the Serine/Arginine-rich (SR) protein family, responds to light and the Target of Rapamycin (TOR) signalling pathway, yet its downstream targets and regulatory impact remain unknown. To identify At-RS31 targets, we applied individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) and RNAcompete assays. Transcriptomic analyses of At-RS31 mutant and overexpressing plants further revealed its effects on alternative splicing. iCLIP identified 4034 At-RS31 binding sites across 1421 genes, enriched in CU-rich and CAGA RNA motifs. Comparative iCLIP and RNAcompete data indicate that the arginine/serine (RS) domain of At-RS31 may influence its binding specificity in planta, underscoring the value of combining in vivo and in vitro approaches. Transcriptomic analysis showed that At-RS31 modulates diverse splicing events, particularly intron retention and exitron splicing, and influences other splicing modulators, acting as a hierarchical regulator. By regulating stress response genes and genes in both TOR and abscisic acid signalling pathways, At-RS31 may help integrate these signals, balancing plant growth with environmental adaptability through alternative splicing.