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Phosphothreonine 218 is required for the function of SR45.1 in regulating flower petal development in Arabidopsis

Xiao-Ning Zhang, Cecilia Mo, Wesley M Garrett, Bret Cooper
Plant signaling & behavior 2014 v.9 no.7 pp. e29134
Arabidopsis thaliana, alanine, alternative splicing, aspartic acid, coprecipitation, corolla, glutamic acid, mass spectrometry, messenger RNA, mutants, peptides, phosphorylation, plant tissues, precipitin tests, quantitative polymerase chain reaction, recombinant fusion proteins, reverse transcriptase polymerase chain reaction, root growth, spliceosomes, threonine, transgenic plants
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.