Using a luciferase reporter assay, our results are the first to demonstrate that the mouse GDNF 3'-UTR has multiple suppressive regions regulate gene expression. In this study, we employed three types of promoters (SV40, CMV and the intrinsic mouse GDNF promoter) and two genes (luciferase and GDNF) to characterize the features of the mouse GDNF 3'-UTR. Among several regions in the mouse GDNF 3'-UTR, we focused on the role of the AREs in the middle region of the 3'-UTR in regulating gene expression in a post-transcriptional manner, such as through mRNA stability, because these particular AREs are higly conserved among eleven different organisms. In our experiments, the C6 cells transfected with an expression construct in which the AU-rich region was immediately downstream of the coding region expressed negligible amounts of mRNA and protein. These results suggest that the suppressive effects of this AU-rich region in the mouse GDNF 3'-UTR are not affected by the coding sequences or promoters. Barreau et al.
have proposed that there are three different classes of AREs [20
], and according to their classification, the AU-rich region in the GDNF 3'-UTR belongs to Class I because it contains the ARE consensus sequences, AUUUA. In our deletion analyses, the reporters in which the AREs were serially deleted gradually lost their suppressive properties, demonstrating that three ARE consensus sequences in the mouse GDNF 3'-UTR play a cooperative role in regulating the amounts of mRNA. However, our results also indicated that this suppressive function does not simply depend on these conserved AREs. The Δ20 construct containing only the core ARE region (+933/+1010) did not exhibit a convincing suppressive effect. The Δ13 construct containing only the short region (+859/+932), just upstream from the ARE region, still exhibited reduced luciferase activity of approximately 50% compared to pGL3pro. It seems that both region adjacent to the AREs might be required to suppress the luciferase activity effectively. Hajarnis et al.
have reported that the GC-rich sequences adjacent to an ARE in the 3'-UTR of phosphoenolpyruvate carboxykinase also function to destabilize the mRNA [21
]. We also demonstrated that the core ARE-deletion (+938/+1014) from entire GDNF 3'-UTR caused an apparent doubling of luciferase activity. It is possible that other suppressive factors recognize currently uncharacterized regions in the mosue GDNF 3'-UTR to exert the full suppressive effect. In contrast to the high conservation of the region around the AREs among 11 species, the distal half of the mouse GDNF 3'-UTR (+1075/+2518) is approximately 85% homologous to only the putative rat GDNF 3'-UTR. Meanwhile, the posterior half of the human GDNF 3'-UTR is highly similar to only the putative Rhesus and chimpanzee sequences. The homology is lower between rodents and primates. As shown in Figures , and , this unconserved region showed a marked suppressive effect on the promoter activity, and the deletion of the core ARE from the Wt and Δ2 constructs recovered the activity to a lesser extent. Therefore, we conclude that in addition to our characteirzed AREs, common and species-specific negative factors cooperatively recognize the concensus sequences in the GDNF 3'-UTR to regulate the expression of the GDNF gene. However, the precise mechanism for the down-regulation of expression by the 3'-UTR remain to be determined.
It has been reported that an ARE in the 3'-UTR regulates the expression of many types of genes, including some cytokines, immediate early genes and trophic factors [18
]. Moreover, many families of RNA-binding proteins that specifically recognize an ARE in several genes have been identified. AUF1 consists of four splicing variants and is reported to destabilize mRNA through an ARE [22
]. In contrast, the ELAV family members, including HuR, HuD, HuB and HuC, are suggested to enhance mRNA stabilization [24
]. We transfected our reporter constructs containing the mouse GDNF 3'-UTR together with the AUF1 splicing variants or the ELAV family members; however, none of the ARE-binding proteins restored luciferase activity in the C6 cells transfected (unpublished data). Some stimuli (e.g
., NGF [26
], GM-CSF [27
], PMA [28
], LPS [29
] and heat shock [30
]) have been reported to stabilize mRNA through the activation of intracellular signaling pathways [26
] and/or the modification of RNA-binding proteins [26
]. We attempted to examine the effects of PMA and LPS, which were previously reported to up-regulate endogenous GDNF mRNA, but neither stimulus inhibited the suppressive effect of an ARE in the mouse GDNF 3'-UTR. As some factors, including poly(A)-binding proteins [33
] and microRNAs [34
], have also been reported to post-transcripionally regulate the amount of mRNA with a 3'-UTR in quantity, we investigated whether PABPc1, a poly(A)-binding protein, affects the suppressive effect of the mouse GDNF 3'-UTR. Our results show that PABPc1 overexpression does not affect this suppressive feature. Therefore, it is still unclear which factors (proteins and/or RNA molecules) participate in GDNF expression via its 3'-UTR. Using miRBase http://www.mirbase.org/
to search for microRNAs that might recognize the ARE and non-ARE regions, we found that some microRNAs (e.g
., mmu-miR-1955-5p and mmu-miR-883a-3p) are predictied to associate with the suppressive regions within the Δ13 and Δ 5 regions, respectively. Thus, further studies on the identification and characterization of negative regulators that destabilize the GDNF mRNA in combination with AREs and other suppressive regions of the GDNF 3'-UTR are required to determine the mehanisms for regulating GDNF expression under pathophysilological conditions.
We previously characterized three distinct mouse GDNF promoters upstream of exons 1, 2 and 3 [35
]. Brodbeck et al.
reported that Six2, a homeobox gene, recognizes its consensus sequence in the mouse GDNF promoter 1 and potentiates its promoter activity [37
]. With the exception of Six2 as a regulator of renal development [38
], none of the transcriptional factors related to neuronal inflammation have been identified, although many inflammatory stimuli are reported to enhance intrinsic GDNF mRNA expression in vivo
and in vitro