Physiological functions and mechanisms on post-transcriptional regulation of RNA-binding proteins in osteoblastic differentiation.

阐明成骨细胞分化的分子机制一直是骨生物学研究的重要课题之一。细胞的分化是由各种转录因子在转录水平上控制的，这些转录因子是通过生化和遗传手段确定的，也是在翻译水平上控制的。在这项研究中，我研究了RNA结合蛋白对成骨细胞分化的转录后调控作用。培养MC3T3-E1细胞和C3H10T1/2细胞，提取总RNA。然后，对RNA结合区的保守序列进行简并RT-PCR。将RT-PCR获得的10多个克隆插入到pGEM-T载体中，用DNA测序仪进行测序。我得到了一个编码核仁素、RNaseZ和几种未知蛋白质的RNA结合特性的cDNA。合成和纯化了它们的重组蛋白，并通过凝胶位移分析证明这些RNA结合蛋白与骨钙素基因非编码区的几个区域结合。将这些表达载体导入未分化间充质细胞，如W-20细胞，发现骨桥蛋白基因在这些细胞中表达上调。这些结果提示，某些与骨钙素基因非编码区相互作用的RNA结合蛋白在成骨细胞分化中起重要作用。

Elucidation of molecular mechanisms controlling differentiation of osteoblasts has been one of the major subjects in bone biology. Differentiation of cells is controlled at the level of transcription by various classes of transcription factors that have been identified through biochemical and genetic means, and the level of translation. In this study, I investigated that a role of post-transcriptional regulation by RNA binding proteins on osteoblastic differentiation. I cultured MC3T3-E1 cells and C3H10T1/2 cells, and then extracted total RNA from these cells. Then, I performed degenerative RT-PCR using primers for conserved sequences of RNA binding domain. More than ten cDNA clones obtained by RT-PCR were inserted to pGEM-T vector and sequenced using DNA sequencer. I got a cDNA encoding RNA-binding properties of nucleolin, RnaseZ and several unknown proteins. Their recombinant proteins were synthesized and purified I also demonstrated that these RNA binding proteins binding to the several regions of UTR of osteocalcin gene by gel shift analyses. After I transfected these cDNA expression plasmids to mesenchymal undifferentiated cells such as W-20 cells, I found that osteopontin mRNA expression up-regulated in these cells. These results suggested that certain RNA binding proteins that interact UTR of osteocalcin gene play important roles for osteoblastic differentiation.