RNA结合蛋白Rbm24调控眼睛晶状体分化的分子机理研究

RNA-binding proteins play important roles in regulating gene expression and tissue-specific alternative splicing events, and are critically involved in organ development and homeostasis. However, only a few tissue-specific splicing factors have been identified at present. Vertebrate lens differentiation requires the degradation of all cytoplasmic organelles (nucleus, Golgi apparatus, endoplasmic reticulum, mitochondria, etc), and the expression of different crystallin proteins, to establish and maintain lens transparency. Genetic mutations of lens-specific regulatory and structural genes lead to defects in lens differentiation and are responsible for congenital and age-related cataracts. The gene encoding a RNA-binding protein, Rbm24, is particularly conserved and is strongly expressed in the lenses of all vertebrates, but its precise function in lens differentiation remains largely unexplored. We found that knockout of Rbm24a in zebrafish markedly delayed the degradation of lens cytoplasmic organelles and specifically down-regulated the expression level of αA-crystallin gene, but not ßB-crystallin and γ-crystallin genes. Cataract formation was observed at 3-day of development. These observations suggest an important role for Rbm24a in the process of lens differentiation. Based on these results, we project to thoroughly analyse the implication and mechanism of Rbm24a in lens differentiation. Since Rbm24 contains only one RNA recognition motif and often operates with other proteins to perform tissue-specific function, we will first identify the interaction partners of Rbm24a in the lenses and determine the target genes regulated by Rbm24a in cooperation with its protein partners. To provide further insight into the tissue-specific function of Rbm24a and the gene regulatory network operating during lens differentiation, we also plan to perform genome-wide identification of Rbm24a-dependent lens tissue-specific alternative splicing events. This work should help to better understand the molecular mechanism underlying lens differentiation and cataract formation. It should also provide insight into the molecular pathways involved in human ocular pathologies.

RNA结合蛋白对调节基因表达和组织特异性可变剪切有至关重要的作用。在脊椎动物眼睛晶状体的分化过程中细胞器降解受阻和纤维化异常是导致先天性白内障的重要原因。Rbm24基因在脊椎动物中高度保守地表达在晶状体中，但它如何调节晶状体的分化还是一个完全空白的研究领域。我们发现在斑马鱼中敲除Rbm24a后使晶状体细胞器降解受阻，作为分子伴侣的晶体基因表达显著下调，导致晶状体透明度异常，说明它对晶状体分化有重要作用。本项目将基于这些发现深入阐明Rbm24a调节晶状体分化的分子机制，开展原创性的工作。进一步的研究目标旨在鉴定与Rbm24a互作的蛋白质以及它们共同调控的靶基因，并在全基因组水平上揭示依赖于Rbm24a的晶状体组织特异性可变剪切事件，明确Rbm24a的作用方式和调节晶状体分化的基因网络。本研究对充分认识晶状体分化和白内障形成的分子机制有重要的理论意义，对预防和治疗眼科疾病有潜在的应用前景。

Rbm24是一个高度保守的RNA结合蛋白，其在脊椎动物的早期胚胎中主要表达在分化过程中的体节、心脏、晶状体及耳泡等组织。已有的研究显示，Rbm24对骨骼肌和心肌细胞的发育有重要作用，而且能够调节一些参与肌细胞分化和结构的基因的可变剪接。然而，目前人们对Rbm24在头部感觉器官发育过程中的作用及其机制则完全不清楚。斑马鱼中有rbm24a和rbm24b两个基因，rbm24a的表达图式与其他脊椎动物的rbm24完全相同，而rbm24b仅在体节和心脏中表达。我们发现rbm24a仅在进入分化状态的晶状体纤维细胞中表达，并首次在斑马鱼中获得了rbm24a突变体。表型分析发现纯合突变体的晶状体发育异常，晶状体纤维细胞不能进行终末分化，导致白内障的形成，说明Rbm24a对晶状体细胞的分化有重要作用。进一步的分子机制研究使我们揭示出Rbm24a在转录后调控中的一个新功能。首先，Rbm24a能够通过其RNA识别基序结合众多的晶状体特异性mRNA，特别是编码各种晶体蛋白的mRNA，其功能缺失导致这些mRNA的多腺甘酸尾显著缩短，翻译效率严重降低，进而阻止晶体蛋白在晶状体纤维细胞中的积累。其次，我们证明Rbm24a通过其碳端与细胞质多腺甘酸化复合物中的成员相结合，说明其可能是调节该复合物的一个新成员。因此，我们的研究结果首次证明Rbm24在脊椎动物中通过调节细胞质多腺甘酸化而影响晶状体分化。此外，我们对rbm24a突变体的拓展研究发现幼鱼具有听力和平衡障碍。分子和细胞生物学分析显示Rbm24a功能缺失导致多个耳聋基因的表达水平降低，内耳和侧线系统的毛细胞形态发生出现异常。该工作首次在内耳发育中鉴别出一个重要的转录后调控因子，为进一步了解毛细胞发育的转录调控机制开辟了新视野。概括起来，本项目达到了预期的研究目标，其创新性成果阐明了Rbm24调控细胞分化的分子机制。