At-RS31 orchestrates hierarchical cross-regulation of splicing factors

The Serine/Arginine (SR)-rich protein At-RS31 is a member of RS subfamily of SR proteins unique to plants. Previous studies have demonstrated that At-RS31 is regulated in response to light and by the Target of Rapamycin (TOR) signaling pathway. Here, we employed individual-nucleotide resolution crosslinking and immunoprecipitation (iCLIP) and RNAcompete assays to identify the targets of At-RS31. The iCLIP analysis revealed 4,034 At-RS31 binding sites across 1,421 genes. We identified distinct RNA-binding motifs enriched in At-RS31 binding sites, including CU-rich and CAGA motifs. Transcriptomic analyses of At-RS31 mutant and overexpressing plants revealed that At-RS31 modulates a broad range of splicing events, particularly affecting intron retention and exitron splicing. It regulates key stress-response and TOR-related genes. At-RS31 also plays a critical role in integrating TOR and abscisic acid signaling pathways , suggesting an important function in coordinating plant growth with environmental responses. By influencing transcription factors and splicing modulators, including other SR proteins, At-RS31 acts as a hierarchical regulator, balancing growth and stress responses through alternative splicing.