Transcriptional And Post-transcriptional Regulation Of Ca²⁺/Calmodulin-dependent Protein Kinase Iγ Expression And Its Role In Neuron Morphogenesis

Ca2+/calmodulin-dependent protein kinase I gamma (CaMKIy), a isoform of the CaMKI family, plays an important role in neuronal morphogenesis. But its function in different stages of neuronal morphogenesis is still not clear. Moreover, the regulatory mechanisms of CaMKIy expression on its transcriptional level and post-transcriptional level are also not revealed. We designed a series of experiments to explore these two aspects and we found the following results.1. To study the effects of CaMKIy on neuronal morphogenesis, we constructed one CaMKIy expression vector and transferred these vectors into mouse primary neurons through electroporation. The results showed that overexpression of the CaMKIy protein in mouse primary neurons can promote dendrites elongation and branch formation in the third and the fourth stage of neuronal morphogenesis.2. Gene expression can be regional or temporal-specifically regulated by enhancers in vivo. To investigate regulatory mechanism on transcriptional level of CaMKIγ, we screened and selected several candidate enhancer sequences by bioinformatic analysis, among which candidate sequence CR2 showed enhancer function. In this study, we selectively cut down sequence CR2 into three fragments, CR2A, CR2B and CR2C respectively. By using in vivo electroporation, we found that CR2C fragment may serve as an enhancer for CaMKIy in the early developmental stage.3. miRNAs involve in regulation of many genes’ expression on post-transcriptional level. To investigate post-transcriptional regulation of CaMKIy we screened and selected several miRNAs which may work on 3’UTR of CaMKIy mRNA. Our results further demonstrated that both miR-135a and miR-135b could inhibit CaMKIy expression by binding CaMKIy mRNA 3’ UTR probably in the seeding sequence region.In summary, our data show that CaMKIy has differential promoting effects on different stages during neuronal morphogenesis. Further we found that CR2C fragment maybe the enhancer that regulated CaMKIy expression at transcription level, while miR-135a and miR-135b negatively regulate CaMKIy expression at the post-transcriptional level.