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

调控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。细胞的分化在转录水平上受到各种类型的转录因子的控制，这些转录因子已经通过生化和遗传手段鉴定出来，并且在翻译水平上受到控制。在本研究中，我研究了RNA结合蛋白转录后调控在成骨细胞分化中的作用。我培养了MC3T3-E1细胞和C3H10T1/2细胞，然后从这些细胞中提取总RNA。然后，我使用引物对RNA结合域的保守序列进行退行性RT-PCR。将RT-PCR获得的10多个cDNA克隆插入到pGEM-T载体上，利用DNA测序仪进行测序。我得到了一个cDNA编码rna结合性质的核仁蛋白，RnaseZ和一些未知的蛋白质。合成和纯化了它们的重组蛋白，并通过凝胶转移分析证明了这些RNA结合蛋白与骨钙素基因UTR的几个区域结合。我将这些cDNA表达质粒转染到W-20等间充质未分化细胞后，发现骨桥蛋白mRNA在这些细胞中表达上调。这些结果表明，与骨钙素基因UTR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.