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

RNA结合蛋白在成骨细胞分化中转录后调控的生理功能和机制。

基本信息

批准号：
13671945
负责人：
TAMURA Masato
金额：
$ 1.92万
依托单位：
HOKKAIDO UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2001
资助国家：
日本
起止时间：
2001 至 2002
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13671945/
关键词：
OSTEOBLAST RNA-BINDING GENE EXPRESSION オステオカルシン転写後調節

项目摘要

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.

阐明控制成骨细胞分化的分子机制一直是骨生物学的主要学科之一。通过生化和遗传手段鉴定出的各种转录因子以及翻译水平，通过各种转录因子来控制细胞的分化。在这项研究中，我调查了RNA结合蛋白在成骨细胞分化中转录后调节的作用。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，我使用引物进行RNA结合结构域的保守序列进行了退化RT-PCR。将通过RT-PCR获得的十个以上的cDNA克隆插入到PGEM-T载体上，并使用DNA Sequencer测序。我得到了一个编码核素，RNasez和几种未知蛋白的RNA结合特性的cDNA。它们的重组蛋白是合成和纯化的，我还证明了这些RNA结合蛋白通过凝胶移位分析结合了骨钙素基因的UTR几个区域。在将这些cDNA表达质粒转染到间充质的未分化细胞（例如W-20细胞）后，我发现这些细胞中骨桥蛋白mRNA表达在这些细胞中上调。这些结果表明，与骨钙素基因相互作用的某些RNA结合蛋白在成骨细胞分化方面起着重要作用。