The Role And Molecular Mechanism Of RNA-binding Protein Rbm24a In Regulating Lens Differentiation

RNA-binding proteins play important role in regulating gene expression and tissue-specific alternative splicing events. Rbm24 is a one of the RNA-binding protein family,with a single RNA-Recognition Motif (RRM) in its N-terminal region. RRMs are the most typical RNA binding modules with a good range of functions in most post-transcriptional regulation processes. In zebrafish,there are two rbm24 genes named rbm24a and rbm24b. rbm24a is expressed both in somites and in heart, and was reported to play an essential role in muscle-specific alternative splicing and sarcomere assembly. In addition, zebrafish rbm24a and other vertebrates rbm24 are particularly conserved and strongly expressed in the lens, but its function in lens differentiation remains unclear. The characteristics of lens epithelial cells (LECs)differentiating into fiber cells are cells cycle exit, cell elongation, establishment of special cell connections, synthesis of crystallins, and losing of the nucleus and other cellular organelles including Golgi apparatus, mitochondria and endoplasmic reticulum, to achieve lens transparency. Defect of crystallin, failure to degrade cytoplasmic organelles during lens cell differentiation and disordered proliferation of lens epithelial cells can affect the fibrosis of lens cells and result in cataract (lens opacities ),which is the leading cause of blindness in the world.We found that knockout of rbm24a using TALENs gene-editing approach resulted in lens differentiation defects including abnormal elongation and denucleation,down-regulated the expression level of ?A-crystallin gene. Cataract formation was observed at 3 days post-fertilization, and homozygous mutants die at 7 days post-fertilization. This results indicate a critical role of rbm24a in lens differentiation.Based on these results, we performed high-throughput RNA-seq analysis to compare the transcriptome between rbm24a mutants and wild-type anterior head to explore the mechanism of rbm24a in lens differentiation. We did not find tissue-specific alternative splicing events but did observe down regulation of crystallin genes expression. Our findings could provide insight into the mechanism of lens differentiation and cataract formation associated with mutation of rbm24a. And it could also provide theoretical basis for the prevention and treatment of cataract.In addition, in order to investigate the role of zebrafish rbm24a and rbm24b genes in the formation and differentiation of somites,we also used CRISPR/Cas9 technology to knock out the rbm24b gene and generated double mutants by crossing.We found that the muscle fibers are disordered and the sarcomere is not obvious in the homozygous double mutants. This work thus provides a foundation to investigate the molecular mechanisms underlying the role of zebrafish rbm24 genes in skeletal musle development..