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

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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.