Dopamine Receptor Drd1 Mrna 3 'utr Of Drd1 Transcriptional Level Expression Of The Role In The Regulation

DRD1 (dopamine receptor D1) is one of dopamine receptors, and is G-protein-coupled receptor constituted by 7 membrane spaning domain. DRD1 widely expresses in nerve sysytem, especially in dorsal caudate putamen, nucleus accumbens septi, optic lemniscus, cortex and amygdaloid nucleus. When DRD1 is actived , it upregulates cAMP ,which then actives protein kinase A, and downstream signal pathways and ion channels. DRD1 has been reported to be important for nerve system's functions, such as movement, study and memory. Abnormal expression of DRD1 has been reported to be associated with many never system diseases, such as drug addiction and Parkinson disease.HuD is a member of ELAVL(embryonic lethal abnormal visual like )/Hu family, which specially express in neuronal system. HuD protein recognises and bind to ARE element by its three RRM motifs, and increases target mRNA stability, involves in transport and translation of target mRNA. HuD plays important roles in outgrow and repair of neurite, regulation of cell cycles of neuron stem cell and neuron precursor cell.ARE elements are important cis-acting elements located in mRNA 3'UTR, which are rich of A and U, and are determinant of RNA instability. According to records of NCBI, Tala et al have assembled all genes which have ARE elements in their 3'UTR to ARED 3.0 database. And we found there was a typical ARE element in DRD1 mRNA's 3'UTR and this ARE is highly conservative in evolution. We found that DRD1 mRNA and HuD mRNA both increased during the differentation of RA induced P19 cell by Real-time PCR. It means HuD protein may recognize the ARE element of DRD1 mRNA 3'UTR and regulat the expression of DRD1.At first, we proved our hypothesis by RNA-chip experiment. We utilized antibody of HuD to capture 'HuD protein-RNA' complex in cytoplasm lysate, and utilized beads with streptavidin to pure this complex, then extracted RNA by guanidnium-Phenol Chloroform, finally we get cDNAs by RT-PCR. We used special PCR primer to amplify special band, and confirm HuD protein bind to DRD1 mRNA in P19 cell. Then we comfirm HuD protein recognize and bind ARE element of DRD1 mRNA 3'UTR by REMSA. This indicated HuD protein bind to DRD1 mRNA by recognize ARE element of DRD1 mRNA 3'UTR.In the dual-luciferase reporter gene experiment in 293ET cell, we found HuD can upregulate DRD1 expression. We inserted wild DRD1 3'UTR to the downstream of the CDS of luciferase. Overexpressed it with HuD protein in 293ET cell, we found the actitivation of luciferase increased. And Real-time PCR revealed the transcripts of wild DRD1 3'UTR increased in the same time. It indicated HuD increase the stability of DRD1 mRNA by recognizing and binding to the ARE element of DRD1 3'UTR, and increase its expression.Finally we found HuD affect the regulation of miR-504 to DRD1. As W.Huang observed in HEK293 cell that has-miR-504 can increase the DRD1, we observed mmu-miR-504 increase DRD1 in 293ET. When we overexpressed miR-504 or HuD alone, the activiation of luciferase increased, but when we overexpressed both HuD protein and miR-504 in 293ET cell, the upregulation of activiation of luciferase was depressed obviously. This is the firs report about the effect between HuD and microRNA.As described above, we found there is an ARE element located in DRD1 3'UTR. HuD protein recognizes and binds to the ARE element, increase the stability of DRD1 mRNA, and upregulate the expression of DRD1. Besids, we found HuD effects the regulation of miR-504 to the mRNA 3'UTR of DRD1.