Methods for Analyzing Alternative Splicing and Its Regulation in Plants: From Gene‐Specific Approaches to Transcriptome‐Wide Studies

Precursor messenger RNA (pre-mRNA) splicing is a fundamental mechanism of gene regulation that influences both mRNA abundance and proteome diversity. In plants, alternative splicing plays a critical role in coordinating development and enabling responses to environmental stress. This process is tightly regulated by the spliceosome and associated splicing factors, which recognize conserved sequence motifs in pre-mRNAs to guide intron removal and exon joining. In this review, we summarize and compare experimental approaches used to analyze both the regulation of alternative splicing and the splicing profiles of genes, spanning from gene-specific assays to transcriptome-wide methods. Gene-specific techniques, such as minigene assays, transient expression systems, electrophoretic mobility shift assays, and isothermal titration calorimetry, provide insights into the molecular interactions between splicing factors and their RNA targets. To identify RNA-binding partners of splicing factors, or splicing factors that interact with a specific RNA, we discuss high-throughput methods that can be applied in vivo and in vitro. By comparing these approaches, we highlight their advantages, limitations, and applications in plant biology. Understanding alternative splicing regulation is essential for deciphering its role in plant adaptation to environmental challenges, with potential implications for crop improvement strategies.