Phosphor-Regulation Induced Pre-mRNA Splicing via a Splicing Activator, SR45, during Flower Development

In eukaryotic cells, most pre-mRNAs contain both introns and exons. The spliceosome assembles at specific sites to produce mature mRNAs. RNA splicing is conserved among all eukaryotic cells and is highly regulated. A splicing activator, Arginine/Serine-rich 45 (SR45), facilitates the initiation of spliceosome assembly. Recent studies showed that a null mutant, sr45-1, exhibited pleiotropic phenotypes, from changes in various developmental stages to altered responses to environmental cues. The pre-mRNA of SR45 encoding gene is alternatively spliced and translated into two protein isoforms differing by seven amino acids. The long form, Isoform 1 (SR45.1), is involved in flower petal development whereas isoform 2 (SR45.2) functions in root growth. Two phosphorylation sites are identified in SR45.1 in inflorescence tissues. Serine 183 (S¹⁸³) is immediately after the RNA Recognition Motif (RRM); threonine 218 (T²¹⁸) is within the alternative fragment. We observed that with S¹⁸³ being phosphorylated, the phosphorylation of T²¹⁸ is required for the normal function of SR45.1. The phosphorylation status of S¹⁸³ and T²¹⁸ affects the predicted SR45.1 protein structure drastically. In addition to previously reported SR45.1-interacting proteins, U1 and U2 snRNPs, we recently found U5 snRNP components in phosphorylated SR45.1 (pSR45.1)-associated proteins. This indicates that SR45.1 may play a role in multiple steps during spliceosome assembly. Using RNA immunoprecipitation (RIP) followed by RNAseq, we identified intron targets of pSR45.1. The current working model is that there may be a crosstalk between S¹⁸³ and T²¹⁸, which determines protein partners in the splicing network and RNA targets that SR45.1 interacts with.