Project description:We identified PRP4 kinase-A (PRP4ka) in a forward genetic screen based on an alternatively-spliced GFP reporter gene in Arabidopsis thaliana (Arabidopsis). Prp4 kinase, which was the first spliceosome-associated kinase shown to regulate splicing in fungi and mammals, has not yet been studied in plants. Analysis of RNA-seq data from the prp4ka mutant revealed widespread perturbations in alternative splicing. A quantitative iTRAQ-based phosphoproteomics investigation of the mutant identified phosphorylation changes in several serine/arginine-rich proteins, which regulate constitutive and alternative splicing, as well as other splicing-related factors. The results demonstrate the importance of PRP4ka in alternative splicing and suggest that PRP4ka may influence alternative splicing patterns by phosphorylating a subset of splicing regulators.
Project description:The epigenetic silencing of the Arabidopsis floral repressor gene FLOWERING LOCUS C (FLC) is induced by a prolonged period of cold and promotes the developmental transition to flowering post-cold. FLC silencing requires the function of VRN1, a non-sequence-specific DNA binding protein. Here, we used Arabidopsis seedlings growing at ambient temperature carrying a FLAG-tagged VRN1 under its endogenous promoter in the vrn1FRI mutant background. We performed FLAG immunoprecipitation followed by mass spectrometry (IP-MS) on crosslinked tissue. The proteomics analysis revealed several proteins that support the role of VRN1 as a chromatin regulator. The identified proteins associated with VRN1 in vivo also support its contribution to gene silencing.
