Characterization of 3'-untranslated region of the mouse GDNF gene
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Glial cell line-derived neurotrophic factor (GDNF) is a potent survival factor for many cell types, and its expression is widespread both within and outside of the nervous system. The regulation of GDNF expression has been extensively investigated but is not fully understood.
Using a luciferase reporter assay, we identified the role of the 3'-untranslated region (3'-UTR) of the mouse GDNF gene in the regulation of gene expression. We focused on a well-conserved A- and T-rich region (approximately 200 bp in length), which is located approximately 1000 bp downstream of the stop codon in exon 4 of the gene and contains three typical AU-rich elements (AREs), AUUUA. Interestingly, these AREs are well conserved in several GDNF genes. By testing reporter constructs containing various regions and lengths of the 3'-UTR fused to the end of the luciferase gene, we demonstrated that the ARE-induced decrease in luciferase activity correlates with the attenuation of the mRNA stability. Furthermore, we found that several regions around the AREs in the 3'-UTR suppressed the luciferase activity. Moreover, the expression level of the GDNF protein was negligible in C6 glioma cells transfected with the ARE-containing GDNF expression vector.
Our study is the first characterization of the possible role of AREs and other suppressive regions in the 3'-UTR in regulating the amounts of GDNF mRNA in C6 cells.
KeywordsLuciferase Activity Enhanced Green Fluorescence Protein Reporter Construct Luciferase mRNA GDNF Gene
List of abbreviations used
glial cell line-derived neurotrophic factor
polymerase chain reaction
enhanced green fluorescence protein.
Glial cell line-derived neurotrophic factor (GDNF) was originally purified from rat B-49-conditioned medium and was characterized as a potent neurotrophic factor for culturing dopaminergic neurons from the developing substantia nigra . GDNF is a distantly related member of the transforming growth factor-β (TGF-β) superfamily , and additional GDNF homologs have also been cloned [3, 4, 5]. GDNF expression is widespread in both the central and peripheral nervous systems, in addition to outside of the nervous system [6, 7, 8, 9]. The targeted disruption of the mouse GDNF gene showed that GDNF plays a critical role in the development of both kidney and enteric neurons during embryogenesis [10, 11]. GDNF possesses multifunctional properties that regulate the development and differentiation of a variety of cell lineages and acts as a neurotrophic factor for specific types of neurons in the nervous system. Accordingly, many investigators have reported the regulation of GDNF mRNA in various types of cells, such as astrocytes, microglial cells and macrophages, both in vitro and in vivo during tissue development and in pathophysiological states, including in response to inflammatory stimuli, ischemic/hypoxic insults and spinal cord injury [12, 13, 14, 15, 16]. However, the precise mechanisms regulating GDNF mRNA expression are not yet fully understood.
For many genes, the promoter and enhancer activities of their 5'-flanking regions and introns have been extensively characterized in the evaluation of gene expression regulation. However, the regulation of mRNA stability has also been demonstrated to play an important role in controlling gene expression . In particular, a sequence rich in adenosine (A) and uridine (U), containing the AU-rich element (ARE), AUUUA, has been identified to regulate expression levels of mRNA. The ARE motif was first identified within the 3'-untranslated regions (3'-UTRs) of mRNAs encoding cytokines , and many genes have been predicted to produce ARE-containing mRNAs [19, 20].
In this study, we focused on the regulatory role of AREs in the 3'-UTR of exon 4 in the mouse GDNF gene. In addition, the ARE-containing region of GDNF exon 4 fused to the end of the luciferase or the mouse GDNF coding region markedly diminished each expression.
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 , 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 . 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 2, 3 and 6, 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, 19, 20]. 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, 23]. In contrast, the ELAV family members, including HuR, HuD, HuB and HuC, are suggested to enhance mRNA stabilization [24, 25, 26, 27]. 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 , GM-CSF , PMA , LPS  and heat shock ) have been reported to stabilize mRNA through the activation of intracellular signaling pathways [26, 27, 28, 29, 30, 31, 32] and/or the modification of RNA-binding proteins . 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  and microRNAs , 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, 36]. Brodbeck et al. reported that Six2, a homeobox gene, recognizes its consensus sequence in the mouse GDNF promoter 1 and potentiates its promoter activity . With the exception of Six2 as a regulator of renal development , 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[12, 13, 14, 15, 16].
Our present study is the first to suggest the possible role of several regions in the 3'-UTR of the mouse GDNF gene in regulating its gene expression. Among these regions, we characterized the suppressive feature of a well-conserved A- and T-rich region (approximately 200 bp in length) in the mouse GDNF 3'-UTR. Based on the well-conserved nucleotide sequences surrounding the AREs among 11 species, the ARE of the human GDNF 3'-UTR is predicted to have a similarly suppressive role. Further characterization of the interaction of this ARE with other suppressive regions in the GDNF 3'-UTR, together with that of the GDNF promoter, will help to clarify the complex regulatory mechanisms of GDNF gene expression.
Construction of plasmids
For the preparation of the reporter constructs containing the 3'-UTR of mouse GDNF, various lengths of the 3'-UTR were amplified by PCR and cloned into the pGL3-Promoter vector (pGL3pro) (Promega) at the Xba I site that is immediately downstream of the luciferase gene. In this study, the nucleotide immediately after the stop codon in exon 4 of the mouse GDNF gene is defined as +1 (Figure 1A). The reporter constructs used in this study are shown in Figure 2. The mouse GDNF 3'-UTR was also cloned into the pGL3-Basic vector containing the mouse GDNF promoter 1 (GDNF pro) . The coding region of mouse GDNF fused with the 3'-UTR was amplified by PCR and then cloned into the pcDNA3.1 vector .
Cell culture and treatment
C6 cells were maintained in Ham's F-10 medium (Invitrogen) supplemented with 3% fetal bovine serum and 7% horse serum. Transfection of each construct used in this study was performed using the Lipofectamine-Plus reagent (Invitrogen) according to the manufacturer's instructions .
Reporter gene assay
The reporter constructs and the pRL-TK vector, an internal control, were transfected into C6 cells in a 48-well plate. Thirty-six hours after transfection, the cells were lysed, and the luciferase activity in each lysate was measured using a Dual-Luciferase assay system (Promega). The reporter activity in each lysate was normalized to the co-transfected Renilla luciferase activity, and the results are shown as the relative luciferase activity.
Western blot analysis
The expression levels of GDNF and EGFP in the cell lysates were estimated by Western blotting, as described previously . Briefly, the transfected cells were lysed with SDS-Laemmli sample buffer [62.5 mM Tris-HCl (pH 6.8), 2% SDS and 10% glycerol], and the protein concentration of each cell lysate was determined using the Protein DC assay kit (Bio-Rad). Equal amounts of each sample were separated by 12.5% SDS-polyacrylamide electrophoresis gels, transferred onto polyvinylidene difluoride membranes (GE Healthcare Bioscience) and identified using a primary antibody against GDNF (Santa Cruz Biotechnology) or EGFP (Roche Biochemicals) and enhanced chemiluminescence (GE Healthcare Biosciences).
Analysis of mRNA stability
The cells were harvested thirty-six hours after co-transfection with each reporter construct and an enhanced green fluorescence protein (EGFP) expression vector (pEGFP-N1) (Clontech) as an internal control. Act-D (5 μg/ml) was added to the cells 1 or 3 h before harvesting the cells, which were lysed with Trizol to extract the total RNA. The total RNA was treated with DNase (NIPPON GENE) for 15 min according to the manufacturer's instructions to degrade the contaminating reporter constructs and estimate the amount of each mRNA. After re-extraction of the treated RNA, the total RNA (0.5 μg) was converted to cDNA by reverse transcription using random ninemers to prime SuperScript III reverse transcriptase (RT) (Invitrogen), as previously described . To estimate the expression level of each mRNA by RT-PCR, the specific cDNAs were mixed and amplified using PCR (Taq PCR kit, Takara). The RT-PCR primers used in this study were as follows: the luciferase sense primer, 5'-GGTGGCTCCCGCTGAATT-3'; the luciferase antisense primer, 5'-GATTTTTCTTGCGTCGAG-3'; the EGFP sense primer, 5'-ACCTACGGCAAGCTGACCCTGAA-3'; the EGFP antisense primer, 5'-CTCCAGCTTGTGCCCCAGGAT-3'; the β-actin sense primer, 5'-TGTATGCCTCTGGTCGTACC-3'; and the β-actin antisense primer, 5'-CCACGTCACACTTCATGATGG-3'. By measuring the remaining RNA to estimate the mRNA stability, we first determined the appropriate number of cycles of amplification for each gene. For the detection of luciferase, EGFP and β-actin mRNAs, the number of cycles of amplification was 28, 25 and 21, respectively. After the amplification of each gene, the products were separated by electrophoresis on 2.0% agarose gels and visualized using ethidium bromide. The fluorescence intensity of each band was scanned and quantified using NIH-Image software [15, 16]. To evaluate the lower amount of luciferase mRNA derived from the pGL3-promoter vector containing the GDNF 3'UTR region, additional cycles of amplification were performed to produce fluorescence intensities in the Δ8-transfected untreated cells (0 h) that were almost similar to those derived from the pGL3pro-transfected cells. The experiments were repeated to confirm the reproducibility.
The results are expressed as the mean ± SD of more than three cultures. The statistical analysis was performed using one way-ANOVA followed by Fischer's PLSD test. A probability of p < 0.01 was considered to be statistically significant.
We would like to thank Shunsuke Toyoda, Hironobu Shitara and Kosuke Oji for their technical assistance.
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