Sequences of the Non-Coding RNA, NTAB, are Contained within the 3′-UTR of Human and Rat EAAT2/GLT-1 Transcripts and Act as Transcriptional Enhancers

Abstract

In the CNS, extracellular glutamate is predominantly cleared by astroglial cells through the high-affinity glutamate transporter subtype, EAAT2/GLT-1. Expression of EAAT2/GLT-1 is perturbed in various acute and chronic brain diseases eventually allowing for the onset of neurotoxic extracellular glutamate concentrations and subsequent excitotoxic neuronal cell death. The idea that glutamate-induced brain damage could be prevented by restoring glutamate homeostasis in the injured brain, spurred considerable interest in identifying the mechanisms controlling EAAT2/GLT-1 expression. Since to date most of this study was done with rat astrocytes, an emerging issue is to whether these findings would also apply to humans. While so far it is known that the promoter region of the EAAT2/GLT-1 gene is strikingly similar in rat and man, little information is available on the function of the EAAT2/GLT-1 3′-UTR in the control of EAAT2/GLT-1 expression in general as well as across both species. We now report on the presence of a homologous sequence within the 3′-UTR of the human and rat EAAT2/GLT-1 gene which we identified as a partial sequence of the putative non-coding RNA, Ntab. We further demonstrate that fragments of Ntab act as enhancers of EAAT2/GLT-1 transcription. Finally, we unravel that partial Ntab sequences are selectively present in the vicinity of the EAAT2/GLT-1 gene in several other mammalians, implying a conserved function of this sequence in the vertebrate CNS.

Electronic supplementary material

The online version of this article (doi:10.1007/s10571-010-9630-9) contains supplementary material, which is available to authorized users.

Introduction

l-glutamate is the major excitatory neurotransmitter in the vertebrate CNS. At high extracellular concentrations, glutamate induces neuronal cell death by a complex mechanism termed excitotoxicity (Yi and Hazell 2006). Extracellular glutamate is predominantly cleared by a family of high-affinity carrier proteins which use the Na⁺/K⁺ electrochemical gradient as a driving force (Kanner 2006). This family currently comprises the five members, glutamate aspartate transporter/excitatory amino acid transporter 1 (GLAST/EAAT1), glutamate transporter-1 (GLT-1)/EAAT2, excitatory amino acid carrier-1 (EAAC1)/EAAT3, EAAT4, and EAAT5 (Torres and Amara 2007). The EAAT2/GLT-1 transporter subtype is predominantly, but not exclusively, expressed by astrocytes (Furness et al. 2008) and accounts for about 90% of total glutamate uptake in most brain areas (Rothstein et al. 1996; Tanaka et al. 1997). EAAT2/GLT-1 expression is perturbed in various acute and chronic brain diseases, including ischemia/hypoxia, traumatic injuries, amyotrophic lateral sclerosis, Alzheimer’s disease, and Huntington’s disease (Sheldon and Robinson 2007). This disease-associated loss or decline of EAAT2/GLT-1 expression eventually leads to increases in extracellular glutamate concentrations and subsequent excitotoxic (secondary) neuronal cell death (Rothstein et al. 1996; Yi and Hazell 2006). The idea that restoring glutamate homeostasis in the injured brain would allow to prevent or minimize glutamate-induced secondary brain damage (Sheldon and Robinson 2007) focussed considerable interest over recent years on defining the mechanisms controlling EAAT2/GLT-1 expression. These studies successfully identified several gene products affecting EAAT2/GLT-1 expression, such as the EGFR ligands, epidermal growth factor (EGF) and transforming growth factor α (TGFα) as well as endothelins (ETs) which either promote or inhibit glial EAAT2/GLT-1 expression, respectively (Figiel et al. 2003; Zelenaia et al. 2000; Rozyczka et al. 2004). Since most of these findings were obtained with rat astrocytes, our interest recently focused on the issue to whether this regulatory network would also apply to human astrocytes. A subsequent comparative analysis of the human and rat EAAT2/GLT-1 promoters revealed that the sequences from both species contain similar cis-regulatory elements responsible for constitutive and regulated EAAT2/GLT-1 expression (Allritz et al. 2010; Su et al. 2003). To complement this issue, we have now compared the structure and function of the 3′-UTR region of the EAAT2/GLT-1 gene in man and rat. We report that in both species the EAAT2/GLT-1 3′-UTR either contains the full-length sequence or a partial sequence of Ntab, which has been originally regarded as a non-coding RNA (French et al. 2001). We further provide evidence that Ntab fragments act as enhancers of EAAT2/GLT-1 transcription.

Methods

Cloning of Human 3′-UTR-Plasmids

For cloning of human 3′-UTR-plasmids, genomic DNA was isolated from human blood using the Spin Blood Mini-Kit (Invitek). 50 ng of genomic DNA was used for PCR reaction. For cloning of fragments pA4 to pA7 of the 3′-UTR of the human EAAT2/GLT-1 gene (see Fig. 1), specific oligonucleotides were designed according to Kim et al. (2003). For cloning fragment pA8, the following oligonucleotides were used: 5′-GGGTGTGTGTGCCTGGTTCTT-3′ (sense), 5′-TGGATTCACAGGTTATTTCCTG-3′ (antisense; accession No. NM_004171.3). PCR reaction was performed with Dream-Taq Polymerase (Fermentas, St. Leon-Rot, Germany) using an annealing temperature of 53.3°C (pA4, 6, 7), 54.8°C (pA5), or 61°C (pA2, 8). Reaction products were separated on 1% agarose gels; products of predicted sizes were excised from the gel and cloned into the pSC-A-cloning vector (Stratagene, La Jolla, CA). After digestion with FseI, clones were separated on 1% agarose gels and subcloned into the FseI site of the pGl3-basic vector (Promega, Madison, WI). The construct additionally contained the human promoter fragment, p-502/-129hum EAAT2/GLT-1, which was recently shown to contain all cis-acting elements essential for constitutive and regulated EAAT2 transcription (Allritz et al. 2010). Nucleotide sequences of PCR products were verified using Big Dye 3.1 sequencing kit (Applied Biosystems, Darmstadt, Germany) and ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems, Darmstadt, Germany).

Fig. 1.

Schematic representation of the EAAT2/GLT-1 3′-UTR of a man and b rat. Whereas the rat genome contains the full-length Ntab sequence, only a partial Ntab sequence (80–4393) is present in the human genome. pA4 to pA8 indicates the location of fragments of the human EAAT2/GLT-1 3′-UTR previously shown to control EAAT2/GLT-1 expression (Kim et al. 2003)

Astroglial Cultures

Cultures, enriched in GFAP-immunoreactive astrocytes (>90%), were established from the cerebral hemispheres and hippocampi of postnatal day (P) 2–3 Sprague–Dawley rats (Charles River, Sulzfeld, Germany) according to a recently established protocol (Figiel et al. 2003). In brief, dissected tissue pieces were trypsinized (0.1%) for 20 min and subsequently dissociated in 90% Hank’s balanced salt solution (Life Technologies) and 10% fetal calf serum (Life Technologies) by trituration through 10 ml plastic pipettes. Pelleted cells (400 g; 5 min) were resuspended in 90% minimum essential medium (MEM; Life Technologies) and 10% fetal calf serum (Life Technologies), seeded into 100 mm culture dishes (hemispheres from three animals per dish; Costar; Cambridge, MA), and coated with poly-d-ornithine (0.1 mg/ml; molecular weight, 30–70 kD; Sigma; Deisenhofen, Germany). Upon reaching confluency, cells were trypsinized and replated. After the second passage, cells were seeded into 100 mm culture dishes (Costar) or 6-well cluster plates and taken into experiment.

Transfection and Luciferase Assay

Primary astrocytes were transiently co-transfected with firefly luciferase reporter plasmids and the control plasmid pGl4.70 (Promega) carrying the renilla luciferase reporter gene using the jetPEI transfection reagent (Qbiogene, Heidelberg; Germany) in the presence of MEM containing 2% FCS. After 24 h, cultured cells were switched to serum-free N2-medium. After 72-h post transfection, firefly luciferase was measured using the dual luciferase assay system according to manufacturer’s instructions (Promega, Madison, WI) in a luminometer (Berthold, Bad Wildbad, Germany). Firefly luciferase activity was routinely normalized to renilla reporter gene expression.

PCR Analysis

For qPCR analysis of EAAT2/GLT-1 and Ntab, total RNA was isolated from primary glial cultures, maintained for 48 h in serum-free N2-medium supplemented with either EGF (100 ng/l, Roche) or endothelin-1 (ET-1, 10 nM, Calbiochem) using the PeqGold isolation kit (PeqLab, Schwalbach, Germany) according to the manufacturer’s instructions. RNA concentration was measured by spectrophotometric absorbance at 260 nm. A total of 5 μg of RNA was reverse transcribed using 200 U/μl of M-MLV (Promega, Madison WI) and 1 μg random hexamer primers (Thermo Electron, Ulm, Germany). RNA levels were quantified by real-time polymerase chain reaction (PCR) using the following primers: EAAT2/GLT-1 (accession number, X67857); sense, 5′-AGG AGC CAA AGC ACC GAA AC-3′; antisense, 5′-CCC GGG AAG GCT ATC AAC AT-3′; product size, 169 bp. Ntab (accession number, AY035551); sense, 5′-GGAGATGTGAAGCAAGCA-3′; antisense, 5′-GGGAATGGGAGAGAAACCAT-3′; product size: 177 bp. ß-actin (internal standard); sense, 5′-CTA CAA TGA GCT GCG TGT GGC-3′; antisense, 5′-CAG GTC CAG ACG CAG GAT GGC-3′; product size, 271 bp (all from Thermo Electron). Thermocycling for each reaction was done in a final volume of 10 μl containing 1 μl of cDNA sample (or standard), 1 μl LightCycler-DNA Master SYBR-Green I (Roche Diagnostics, Mannheim, Germany), MgCl₂ (2.5 mM), and sense and antisense primers (5 pmol each). The thermal cycling conditions were 95°C for 90 s followed by 45 cycles of 55°C for 5 s and 72°C for 20 s. Data were collected using the LightCycler Instrument (Roche Diagnostics). To confirm the specificity of the amplified products, melting curves were performed at the end of the amplification by cooling samples at 20°C/s to 65°C for 15 s and then increasing temperature to 95°C at 0.1°C/s with continuous fluorescence measurement. For generation of standard curves, PCR products were purified using QIAquick (Qiagen, Hilden, Germany) spin columns and serially diluted in double-distilled water. The second derivative maximum method as provided by the manufacturer’s software was applied to determine the maximum acceleration of the amplification process, and the respective cycle number was used as a crossing-point value. The concentrations of unknown samples were determined by setting their crossing-points to the standard curve and were normalized to ß-actin.

Northern Blot Analysis

RNA was extracted from the cortex of P1 rats using TriFast (PeqLab), separated on formaldehyde-agarose gels, and transferred onto Zeta-Probe membranes (Biorad). Northern blot hybridization was performed using a P32-labeled Ntab probe corresponding to nucleotides 301–1266 (accession no, AY035551) as well as a P32-labeled rat EAAT2/GLT-1 probe corresponding to nucleotides 811–1671 (accession no, X67857) in hybridization solution (5× SSC, 5× Denhardt’s, 0.2% SDS, and 100 μg/ml of single-stranded salmon sperm DNA) overnight at 68°C. After washing, signal was detected using a phosphoimager system (Fuji). GAPDH mRNA served as a loading control.

Statistics

Data represent mean + SD from at least three independent experiments. Data were subjected to one way analysis of variance (ANOVA) followed by pairwise multiple comparison procedures (Student–Newman–Keuls method). Differences with P < 0.05 were considered significant.

Results

Comparison of the previously cloned 3′-UTR region of the human EAAT2/GLT-1 transcript (Kim et al. 2003; accession no. AY066021/NM_004171.3) with the rat genome showed that the distal part of the 3′-UTR shows a higher homology (71–77%) to human full-length EAAT2/GLT1 than the proximal part (64–74%). This highly homologous stretch is located roughly 5000 bp downstream of the open reading frame of the EAAT2 gene in man (NM_004171.3) and rat (Figs. 1, 2). When we analyzed whether the sequence belongs to an expressed gene, we surprisingly found that the homologous stretch represents a partial sequence of Ntab, a putative non-coding RNA comprising 4400 bp which has been previously identified in rat brain (French et al. 2001; Fig. 1). Genome wide BLAST further revealed that only the rat genome contains the full-length Ntab sequence, whereas similar to man the genome of other mammalians only contain partial Ntab sequences (Figs. 1, 2). Notably, in all vertebrate species, these Ntab sequences are exclusively located 4700–5200 bp down-stream of the EAAT2/GLT-1 ORF (Fig. 2).

Fig. 2.

Schematic representation of Ntab sequences conserved in various mammalian species. Comparison of Ntab (AY035551) with genome assemblies (NCBI, genomic BLAST databases) of various mammals showed conservation of long stretches of Ntab sequences. In the various species, Ntab sequences are located 4710–5211 bp downstream of the EAAT2/GLT-1 ORF. QC query coverage

In man, the partial Ntab sequence is not only part of the 3′-UTR of the EAAT2/GLT-1 transcript, but in addition covers several 3′-UTR fragments which have been previously shown to enhance reporter gene expression (Kim et al. 2003; Fig. 1). Since evidence for a role of these 3′-UTR fragments in the control of EAAT2/GLT-1 gene expression was originally obtained using a reporter gene assay under control of the CMV promoter (Kim et al. 2003), we reanalyzed the effects of these fragments using a reporter gene construct under control of the human EAAT2/GLT-1 promoter (Fig. 3a). In these experiments, we further focused on those 3′-UTR fragments which encompassed Ntab sequences (pA4, pA5, pA6, pA7, pA8; see Fig. 1). We found that all 3′-UTR fragments increased reporter gene (LUC) activity in primary astrocytes of the rat hippocampus (Fig. 3b). In astrocytes of the rat cerebral cortex, increases occurred with pA6 and pA8 (Fig. 3c). In addition, LUC activity tended to increase with pA4 and pA5 although these increases were statistically not significant. In contrast, increases in LUC activity were virtually absent with pA7. Moreover, hippocampal astrocytes exhibited the highest increases in LUC activity with the pA6-EAAT2 promoter construct whereas in cortical astrocytes the highest increases occurred with the pA8-EAAT2 promoter construct. Notably, cortical and hippocampal astrocytes transfected with the reporter construct under control of the EAAT2/GLT-1 promoter exhibited a similar ratio of firefly/renilla luciferase activity (cortical astrocytes 19.6 ±10.5; hippocampal astrocytes 19.2 ± 0.8; n = 3). We consider these findings as a hint that the differential effects seen with the various fragments in cortical and hippocampal astrocytes do not necessarily relate to brain region-specific differences in the promoter activity per se. Together, these findings confirm that fragments of the human EAAT2/GLT-1 3′-UTR enhance EAAT2/GLT-1 transcription. In addition, the findings point to a brain region-specific function of the 3′-UTR fragments. Contrasting to the previous demonstration that pA4 under control of the CMV promoter suppresses reporter gene expression (Kim et al. 2003), these findings further demonstrate enhanced reporter gene activity with pA4 under control of the human EAAT2/GLT-1 promoter.

Fig. 3.

Effects of human EAAT2/GLT-1 3′-UTR fragments on reporter gene activity. a Schematic representation of luciferase reporter gene plasmids containing the human EAAT2/GLT-1 promoter (p-502/-129hum) and fragments of the human EAAT2/GLT-1 3′-UTR (pAx). b, c Luciferase activity in astrocytes cultured from b rat hippocampus and c rat cortex. Cells were transiently co-transfected with firefly luciferase reporter plasmids and control plasmids carrying the renilla luciferase reporter gene. After 72 h, luciferase activity was determined using the dual luciferase assay system as described under Methods. Luciferase activity present in astrocytes transfected with a plasmid containing the EAAT2/GLT-1 promoter sequence, p-502/-129hum, was set to 1. **P < 0.001; *P < 0.01

The data presently available point to Ntab as a non-coding RNA (French et al. 2001). To assess whether Ntab in fact represents an independent transcript in rat, we performed Northern analysis. Since labeleling of poly(A) + RNA isolated from cultured rat astrocytes with specific probes for Ntab produced extremely faint bands, we probed poly(A) + RNA isolated from the cortex of postnatal day 1 rats; this corresponds to the age of the animals used for setting up astrocytic cultures. This approach allowed labeling of a band of about 11 kb as well as of a second band of about 3–4 kb (Fig. 4). It has been recently suggested that this latter band represents the full-length Ntab sequence (French et al. 2001). Surprisingly, the use of selective probes for EAAT2/GLT-1 mRNA resulted in labeling of a single band of about 11 kb (Chen et al. 2002; Schmitt et al. 2002), which seems to be identical to the band labeled by Ntab probes (Fig. 4). To further verify that Ntab is part of the EAAT2/GLT-mRNA, we analyzed the effects of EGF and ET-1, which conversely regulate EAAT2/GLT-1 expression (Figiel et al. 2003; Zelenaia et al. 2000; Rozyczka et al. 2004), on Ntab mRNA levels in cultured cortical astrocytes by quantitative PCR analysis. Corroborating our previous findings (Figiel et al. 2003; Rozyczka et al. 2004), EGF increased astrocytic EAAT2/GLT-1 mRNA levels, whereas ET-1 decreased EAAT2/GLT-1 transcript levels (Fig. 5). Again this was accompanied by an increase and a decrease in Ntab transcripts with EGF and ET-1, respectively (Fig. 5). Since the findings with EGF could be biased by the involvement of Ntab sequences in the stimulatory influences of EGF on EAAT2/GLT-1 expression, we additionally analyzed reporter gene activity of cortical astrocytes transfected with the various 3′-UTR fragments following treatment with EGF. None of the fragments increased reporter gene activity beyond that detectable in (control) astrocytes solely transfected with the EAA2/GLT-1 promoter (Supplementary Fig. 1), hence, suggesting that the stimulatory influences of EGF are not mediated by Ntab sequences. Collectively, these observations establish that Ntab is contained within the 3′-UTR of the rat EAAT2/GLT-1 transcript.

Fig. 4.

Ntab is contained within the 3′-UTR of the rat EAAT2/GLT-1 transcript. Northern analysis of mRNA isolated from the cortex of P1 rats allowed to demonstrate that the Ntab sequence is selectively present in two transcripts with sizes of 3–4 and 11 kb. The use of EAAT2/GLT-1 probes further demonstrated that the 11 kb transcript represents EAAT2/GLT-1 mRNA

Fig. 5.

Coordinated modulation of astrocytic EAAT2/GLT-1 and Ntab transcript levels by EGF and ET-1. Cultures of cortical astrocytes were maintained with EGF (100 ng/ml) or ET-1 (10 nM) for 48 h and subsequently analyzed for EAAT2/GLT-1 and Ntab mRNA levels by quantitative PCR. EGF resulted in a co-ordinated increase of EAAT2/GLT-1 and Ntab transcript levels whereas levels of both transcripts decreased with ET-1. a, P < 0.01, EGF versus control; b, P < 0.01, EGF versus ET-1

Discussion

The role of the 3′-UTR in the control of EAAT2/GLT-1 expression under physiological and pathophysiological conditions is just beginning to emerge. This issue is especially intrigued by the fact that in both man and rat the 3′-UTR of the EAAT2/GLT-1 transcript is extremely long (Kim et al. 2003, and this study), thus, hinting to complex regulatory functions. Along this line, a previous work (Kim et al. 2003) already noted the presence of a variety of regulatory elements within the human EAAT2/GLT-1 3′-UTR, including enhancer elements, AU-rich elements, which are known to define the cytoplasmatic half-life of mRNA molecules (Barreau et al. 2006; Clark et al. 2009) as well as putative polyadenylation sites, essential for nuclear mRNA export, translation, and mRNA decay (Mangus et al. 2003; Andreassi and Riccio 2009). In an attempt to further validate the use of rat astrocytes as a model system for analyzing the regulation of human EAAT2/GLT-1 expression (Allritz et al. 2010), we have now compared the 3′-UTR of the human and rat EAAT2/GLT-1 transcripts. We found that the 3′-UTR of human and rat EAAT2/GLT-1 transcripts comprise a homologous sequence which we identified as Ntab. Notably, only the rat EAAT2/GLT-1 transcript contained the full-length Ntab sequence whereas the human transcript contained only a partial Ntab sequence.

Ntab was originally identified as a putative non-coding RNA selectively expressed in the rat CNS (French et al. 2001). Several of our findings now establish that the full-length Ntab sequence is part of the 3′-UTR of the rat EAAT2/GLT-1 transcript. (1) Northern blot analysis resulted in labeling of an identical band of about 11 kb when hybridized with probes for Ntab and EAAT2/GLT-1. (2) Treatment of cultured cortical astrocytes with EGF, an established stimulator of EAAT2/GLT-1 expression (Figiel et al. 2003; Zelenaia et al. 2000), resulted in a corresponding increase in EAAT2/GLT-1 and Ntab mRNA levels, whereas treatment with ET-1, an established inhibitor of EAAT2/GLT-1 expression (Rozyczka et al. 2004), resulted in a corresponding decrease in the levels of both transcripts. We further demonstrate that the partial Ntab sequence present in the human EAAT2/GLT-1 3’-UTR acts as an enhancer of EAAT2/GLT-1 expression. A previous study already pointed to the existence of various fragments within the human EAAT2/GLT-1 3′-UTR which enhance EAAT2/GLT-1 transcription (Kim et al. 2003), but failed to recognize that some of these fragments, namely pA4, pA5, pA6 pA7 and pA8, represent Ntab sequences. We also found that the various fragments have differential effects on reporter gene expression when assayed in cortical and hippocampal astrocytes, implying that depending on the part of the CNS or the cell type, the 3′-UTR is targeted by different regulatory factors. This conclusion is also in line with the previous demonstration that these fragments differentially affect reporter gene expression in primary human astrocytes and human glioma cells (Kim et al. 2003). Contradicting the previous demonstration that the pA4 fragment represses EAAT2/GLT-1 in human astrocytes and glioma cells (Kim et al. 2003), we found enhanced EAAT2/GLT-1 expression with this fragment in rat astrocytes. We are currently unable to discern whether this diverse finding relates to species or to the fact that Kim et al. (2003) used a CMV promoter for reporter gene analysis whereas we used the human EAAT2/GLT-1 promoter (Allritz et al. 2010).

Intriguingly, Northern blot analysis also demonstrated that in addition to the 11 kb transcript, Ntab probes, but not EAAT2/GLT-1 probes, hybridize to a second band of about 3–4 kb, which was previously shown to represent Ntab (French et al. 2001). Whether this indicates that Ntab is bifunctional in rats, in terms that in addition to representing an enhancer element within the EAAT2/GLT-1 3’-UTR, it would also act as a microRNA remains to be established.

Genome wide analysis finally demonstrated that Ntab core sequences as present in humans are also selectively located in the vicinity of the EAAT2/GLT-1 gene in most other vertebrates, implying that the regulatory function of Ntab on EAAT2/GLT-1 expression might be conserved across vertebrate species.

Electronic Supplementary Material

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