FUG1 SUMO protease regulates splicing via U2AF35B splicingfactor in Arabidopsis thaliana

AbstractBackground &amp; Aim: Alternative splicing (AS) enables eukaryotic genes to generate multiple transcript isoforms, playing a crucial role in plant development and environmental adaptation. Precise regulation of AS is governed not only by core splicing factors but also by post-translational modifications such as SUMOylation. Despite their established roles in animals, the functional relevance of SUMOylation in plant splicing remains underexplored. The SUMO protease FOURTH ULP GENE CLASS 1 (FUG1) has been previously linked to chromatin silencing but was recently found to interact with the splicing factor U2AF35B, which recognizes 3′ splice sites during spliceosome assembly. This study aimed to elucidate the role of FUG1 gene in splicing regulation and its downstream developmental effects in Arabidopsis thaliana.Methodology: In the current study, wild type Bur-0 Arabidopsis thaliana accession and its fug1 mutants, and a U2AF35B knockdown, fug1_35S::amiR-U2AF35B double mutant were used. Plants were grown at 23°C and 27°C short day (SD) conditions to characterize the phenotypes. RNA sequencing (RNA-seq) based transcriptome profiling of Bur-0 and fug1 accessions was performed to identify splicing variations using the SpliSER bioinformatic pipeline. Splicing events predicted by SpliSER were first examined using IGV visualization and sequence alignments, with a focus on intron retention and isoform changes. Selected candidate genes were subsequently validated by RT-PCR and gel electrophoresis. Phenotypic traits such as hypocotyl length, petiole length, and flowering time were documented and compared across genotypes and temperature conditions to determine developmental consequences of splicing disruption.Results: Loss of FUG1 resulted in consistent intron retention and altered isoform ratios in several developmentally important genes, including AT1G21350 (a thioredoxin-like gene) and AT3G56440 (an autophagy regulator). These defects were suppressed in the fug1_35S::amiR-U2AF35B background, indicating that FUG1 regulates splicing via U2AF35B. At 27°C, fug1 mutants exhibited early flowering, a phenotype fully suppressed by U2AF35B knockdown, suggesting an epistatic relationship. AT1G21350 mis-splicing was linked to redox imbalance, which may disrupt circadian and photoperiodic pathways that control flowering. Hypocotyl growth defects at 23°C and temperature-sensitive flowering responses were also regulated by the FUG1–U2AF35B axis, reinforcing its developmental relevance. A regulatory model is proposed wherein FUG1 maintains U2AF35B in a deSUMOylated, functional state; loss of FUG1 leads to U2AF35B hyperSUMOylation, mis-splicing of redox-related genes, and premature floral induction under high temperatures.Conclusion: This study uncovers a novel post-translational regulatory module in which FUG1 controls splicing fidelity by modulating U2AF35B SUMOylation. The FUG1–U2AF35B axis links splicing control to redox signalling and flowering time, particularly under elevated temperatures. While genetic and phenotypic data strongly support this model, future work involving co-immunoprecipitation, SUMOylation assays, and transcriptome-wide splicing rescue analysis will be essential to validate the biochemical underpinnings. These findings provide new insight into how SUMO-mediated splicing control enables plants to fine-tune developmental transitions in response to environmental cues, offering potential strategies for improving stress-resilient crop design.<br>