RNA结合蛋白Samd4调控骨骼发育的机理研究

Skelton development and remodeling are critical for human bodies. The defects of bone development result in different genetic diseases including craniofacial abnormalities. The defects of bone remodeling contribute to the pathomechanisms of skeletal disorders, such as osteoporosis. The mRNA products transcribed in nuclei are translated to functional protein after being modified. At the same time, mRNAs could be degraded along with translational repression, in which a list of micro-RNAs and RNA binding proteins are reported to be involved in. Several miRNAs have been suggested to play important roles during the bone development, meanwhile little is known about whether RNA binding protein acts on this process. We found the insertion of transposon in mouse Samd4 gene caused shorten bone and reduced bone mass, implying that Samd4 could regulate bone development. However, the molecular mechanism of Samd4 in regulation of bone development is unknown. We propose to further analyze the skeleton phenotype of Samd4 knockout mice and extend these studies to identify the direct mRNA target of Smad4 by a combination of biochemical, molecular and cellular biological approaches. The study from Samd4 mice will broaden our knowledge about skeleton development and related diseases.

骨的发育与重塑是人体重要的生理活动。骨发育异常会导致颅面部发育畸形等先天性疾病，骨重塑异常会导致骨质疏松症等疾病。生命活动中，转录产生的mRNA经过一定修饰后能翻译形成功能性蛋白质。蛋白质翻译受到多种因素的调控，例如mRNA的降解等，这一过程受到miRNAs和RNA结合蛋白的影响。目前已经发现多种miRNAs能够调控骨的发育，但是RNA结合蛋白通过控制mRNA翻译而调控骨发育却鲜有报道。我们研究发现RNA结合蛋白Samd4突变小鼠的骨骼发育异常、骨量下降，提示Samd4蛋白能够调控骨骼发育。本项目拟进一步分析Samd4突变小鼠骨发育异常表型，利用生物化学、分子生物学和离体骨细胞培养等手段分析Samd4敲除对骨发育的影响；同时，利用RNA IP等手段，鉴定Samd4调节的靶基因，阐明Samd4及其靶基因在骨骼发育过程中的调节机理，丰富骨骼发育以及相关疾病的调控网络。

广义上的表观遗传-即基因序列不改变的前提下蛋白质水平发生改变，包括DNA修饰、组蛋白修饰以及mRNA转录后修饰。其中mRNA稳定性及蛋白质翻译活性，也属于在许多生理和病理过程中起到重要作用，但目前为止尚无在骨系统中的研究。通过筛选突变体库，我们发现一个RNA结合蛋白SAMD4能够控制小鼠中胚层间质干细胞分化，影响软骨、骨、肌肉和脂肪组织形成。通过RNA IP测序实验，我们发现多个SAMD4能够结合和抑制多个mRNA，导致编码蛋白水平降低，其中变化最剧烈的是Mig6。通过密度梯度离心、和荧光报告基因实验，我们证明了SAMD4能在体内/体外结合Mig6的mRNA，并阻止其与核糖体的结合，从而抑制翻译。通过软骨和成骨细胞的体外培养与分化实验，我们除了验证Mig6已报道的抑制软骨分化功能，同样证明了Mig6能够抑制成骨作用。总之我们首次报道了mRNA翻译导致的蛋白水平变化在骨形成、骨代谢以及相关疾病例如骨质疏松等起到重要作用，首次证明了SAMD4促进成软骨、和成骨作用，以及下游基因Mig6对软骨和成骨的抑制作用，为骨疾病比如骨关节炎、骨质疏松症等相关药物研发，提供了新的靶点。