An Investigation of the Interaction Between Two Phosphorylation Sites in a Splicing Activator SR45.1 in Arabidopsis thaliana

In Arabidopsis thaliana, SR45 is known as a pre-mRNA splicing activator that is most likely to be an ortholog of human protein, RNPS1. RNPS1 is involved in the activation of pre-mRNA splicing during post-transcriptional regulations. SR45 protein has 2 RS domains, rich in serine (S) and arginine (R) dipeptides, that flank one RNA recognition motif (RRM). The SR45 protein encoding gene undergoes alternative splicing to create two isoforms that have been studied in the past. Isoform 1 (SR45.1), consisting of 7 more amino acids than isoform 2 (SR45.2), is involved in flower petal development; SR45.2 regulates root growth. Mass spectrometry suggested two phosphorylation events around S¹⁸³ and T²¹⁸ residues in SR45.1 protein from inflorescence. A mutation at either S¹⁸³ or T²¹⁸ showed narrower flower petals, which is seen in the sr45-1 mutant. Our purpose is to investigate the interaction between these two phosphorylation sites. Four combinations of mutations were created: S183D, S183A + T218D, S183D + T218D, and S183A + T218A. To enable studies in their affinity to RNA targets, these four SR45.1 mutant variants were expressed in insect cell lines using a Bac-to-Bac Baculovirus Expression System. Currently, we are in the process of creating the transgenic plants expressing these four SR45.1 variants in the sr45-1 mutant plants, separately. Combining both in vitro and in vivo studies, the goal is to achieve a better understanding in how SR45.1 regulates alternative splicing to support normal flower development.