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.

Institute of Plant Breeding and Genetic Resources ELGO DIMITRA Thermi Greece

Institute of Plant Genetics and Biotechnology Plant Science and Biodiversity Centre Slovak Academy of Sciences Nitra Slovakia

Mendel Center for Plant Genomics and Proteomics CEITEC MU Central European Institute of Technology Masaryk University Brno Czech Republic

Molecular and Cell Biology of Plants Institute of Molecular Biosciences Goethe University Frankfurt Frankfurt am Main Germany

National Centre for Biomolecular Research Faculty of Science Masaryk University Brno Czech Republic

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