Functional study of the Ser/Arg-rich splicing factor SRSF5a during zebrafish embryonic development

Nuclear pre-mRNA splicing is a key process regulating gene expression in eukaryotes. Splicing consists in the removal of introns and the joining of exons within a dynamic macromolecular complex called the spliceosome, which consist of five small nuclear ribonucleoproteins (snRNPs) and numerous non snRNPs proteins (1). Amongst these non snRNPs proteins, the SR proteins family constituted an important group of splicing factors that are involved in constitutive and alternative splicing (2,3). SR proteins are structurally related as they are characterized by one or two RNA-recognition motifs (RRMs) in N-ter and a C-terminal domain enriched in dipeptide Ser/Arg. Phylogenetic inference using the RRM domain allowed us to identify 13 encoding genes for SR proteins in the vertebrate model organism, Danio rerio. The Zebrafish is increasingly recognized as a powerful model for the study of vertebrate embryonic development in a physiological context. The roles of SR splicing factors during animal cell differentiation and development are largely unknown. The aim of the present research is to investigate SR proteins functions during zebrafish development by using molecular and genetic approaches. In this study, we investigated the role of the SR splicing factor SRSF5a. The expression profile was determined by in situ hybridization at 24, 48 and 72 hours post-fertilization and showed SRSF5a expression mainly in brain, retina and pharyngeal arches at these stages. Furthermore, SRSF5a knock-down by morpholinos microinjection strongly suggests an important role of this specific splicing factor during eyes and brain development. In order to gain insight into the molecular function of SRSF5a, we analysed control and morphant transcriptomes using high throughput RNA sequencing. Finally, we use a complementary approach to morpholinos and generate SRSF5a mutant fishes using TALENs (Tal effector nucleases)(4,5).
1. Wahl MC, Will CL, & Luhrmann R (2009) The spliceosome: design principles of a dynamic RNP machine. Cell 136(4):701-718.
2. Long JC & Caceres JF (2009) The SR protein family of splicing factors: master regulators of gene expression. Biochem J 417(1):15-27.
3. Graveley BR (2000) Sorting out the complexity of SR protein functions. RNA 6(9):1197-1211.
4. Moore et al. (2012) Improved Somatic Mutagenesis in Zebrafish Using Transcription Activator-Like Effector Nucleases (TALENs). Plos One 120(1):1-12.
5. Cade et al (2012) Highly efficient generation of heritable zebrafish gene mutations using homo- and heterodimeric TALENs. Nucleic acid research Vol 40, No 16:8001-8010.