title: Phosphothreonine 218 is required for the function of SR45.1 in regulating flower petal development in Arabidopsis.
abstract: RNA splicing is crucial to the production of mature mRNAs (mRNA). In Arabidopsis thaliana, the protein Arginine/Serine-rich 45 (SR45) acts as an RNA splicing activator and initiates the spliceosome assembly. SR45 is alternatively spliced into 2 isoforms. Isoform 1 (SR45.1) plays an important role in the flower petal development whereas isoform 2 (SR45.2) is important for root growth. In this study, we used immunoprecipitation to isolate an SR45.1-GFP fusion protein from transgenic plants complementing a null mutant, sr45-1. Mass spectrometry suggested a single phosphorylation event in a peptide from the alternatively spliced region unique to SR45.1. Substituting alanine for threonine 218, a candidate site for phosphorylation, did not complement the sr45-1 mutant with narrow flower petals whereas substituting aspartic acid or glutamic acid for threonine 218 did complement the sr45-1 mutant. Mass spectrometry also revealed that other proteins involved in the spliceosome co-precipitated with SR45.1, and RT-qPCR revealed that phosphorylation of threonine 218 promotes the function of SR45.1 in promoting the constitutive splicing of SR30 mRNA. This is the first demonstration of a specific phosphorylation site that differentially regulates the function of a plant splicing activator in physiologically and morphologically distinct plant tissues.
authors.affiliation: a Department of Biology; Saint Bonaventure University; Saint Bonaventure, NY USA.
authors.forename: Xiao-Ning
authors.initials: XN
authors.lastname: Zhang
authors.forename: Cecilia
authors.initials: C
authors.lastname: Mo
authors.forename: Wesley M
authors.initials: WM
authors.lastname: Garrett
authors.forename: Bret
authors.initials: B
authors.lastname: Cooper
mesh_terms: D017398:Alternative Splicing; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000378:metabolism; D017360:Arabidopsis / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism; D035264:Flowers / Q000254:growth & development / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D010769:Phosphothreonine / Q000378:metabolism*; D030821:Plants, Genetically Modified; D020033:Protein Isoforms; D012326:RNA Splicing; D012333:RNA, Messenger / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D000068103:Serine-Arginine Splicing Factors; D017381:Spliceosomes / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D000596:Amino Acids; D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D009687:Nuclear Proteins; D010455:Peptides; D020033:Protein Isoforms; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C581090:SR30 protein, Arabidopsis; C483303:SR45 protein, Arabidopsis; D010769:Phosphothreonine; D000068103:Serine-Arginine Splicing Factors; D012694:Serine
keywords: Co-immunoprecipitation; U5 small nuclear ribonucleoprotein; flower petal development; protein phosphorylation; regulation of RNA splicing