Next-Generation Mapping of the ACINUS-Mediated Alternative Splicing Machinery and Its Regulation by O-glycosylation in Arabidopsis

Alternative splicing (AS) is a key mechanism of gene regulation, but the full repertoire of proteins involved and the regulatory mechanisms governing this process remains poorly understood. Using TurboID-based proximity labeling coupled with mass spectrometry (PL-MS), we comprehensively mapped the Arabidopsis AS machinery, focusing on the evolutionarily conserved splicing factor ACINUS, its paralog PININ, and the stable interactor SR45. Our analysis identified a high-confidence list of 298 components, including both established and novel interactors, providing strong evidence that alternative splicing is coupled to transcription and that multiple RNA processing steps occur simultaneously in plants. Selected interactors were validated through AS readouts and phenotypic analysis. Furthermore, a systematic evaluation of O-glycosylation double mutants revealed that SECRET AGENT (O-GlcNAc transferase) and SPINDLY (O-fucose transferase) modulate AS through both ACINUS-dependent and -independent pathways. We used whole seedlings of Arabidopsis thaliana to explore the transcriptomic and alternative splicing changes between WT, acinus pinin, spy-4, Dex inducible SEC RNAi spy-4 lines ( mock- and Dex-treated). Four biological replicates were included for each sample type.