©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Gene Structure of the Murine N-Methyl

D

-Aspartate Receptor Subunit NR2C (*)

(Received for publication, July 27, 1994; and in revised form, September 15, 1994)

Bettina Suchanek (§) Peter H. Seeburg Rolf Sprengel (¶)

From the Laboratory of Molecular Neuroendocrinology, Center for Molecular Biology (ZMBH), University of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The murine N-methyl D-aspartate receptor subunit NR2C (-3) is encoded by a unique gene composed of 12 translated and three 5`-untranslated exons that spread over 20 kilobases of genomic sequence. The GC-rich promoter that lacks TATA- and CAAT-positioning elements has two transcriptional start sites separated by 18 base pairs. One of these sites is located in a conserved initiator motif and, together with the first four exons, specifies the 5`-untranslated sequence of 772 nucleotides. In this sequence, two alternative splice variants were detected that show identical expression patterns in adult mouse brain. Comparison of intron positions in genes encoding different members of the glutamate receptor family confirms a close evolutionary relationship of the NR2C and NMDAR1 subunit genes.


INTRODUCTION

In the vertebrate central nervous system, ionotropic glutamate receptors mediate the fast synaptic action of the major excitatory neurotransmitter, L-glutamate. Traditionally, these receptors have been categorized into functionally different types: alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors, high-affinity kainate receptors, and N-methyl D-aspartate (NMDA) (^1)receptors (reviewed in (3, 4, 5, 6, 7, 8) ). The alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptors mediate the fast component of excitatory postsynaptic currents, whereas the slow component is contributed by the NMDA receptors. The NMDA receptor with its characteristic voltage-dependent Mg block can be considered as a coincidence detector of pre- and postsynaptic activity, and its activation is thought to trigger synaptic modulation underlying memory formation. Excessive glutamate stimulation can induce neuronal cell death due to the high Ca permeability of this channel.

Similar to other ligand-gated ion channels, the ionotropic glutamate receptors are composed of subunits. For the NMDA receptor, two types of subunits have been molecularly characterized, and members of both are needed to form an active ion channel. The NMDAR1 (NR1) subunit occurs in virtually every neuron and is likely to be a constituent of all NMDA receptor subtypes. Four distinct NR2 subunits (NR2A to NR2D for rat; 1 to 4 for mouse) have been isolated(9, 10, 11, 12) . Their expression is developmentally controlled and restricted to specific brain areas(13) . In NR1/NR2 heteromeric channel configurations, the NR2 subunits determine the subtype-specific channel characteristics(10, 11, 13, 14, 15) . The NR1 and NR2 subunits are little related in primary sequence, but are conserved in their general design. They are expressed as large precursor polypeptides with an N-terminal signal peptide. The mature subunits are predicted to contain four hydrophobic transmembrane sequences (M1 to M4) and a discontinuous agonist-binding region(14) . The NR2 subunits contain a uniquely large C terminus of unknown function.

Most members of the ionotropic glutamate receptor family occur as molecularly different variants post-transcriptionally modified by alternative splicing and/or RNA editing. Thus, the NR1 subunit exists in eight different molecular forms(16, 17) . No splice variants have been reported for the NR2 subunits. Here we analyze the gene structure of the murine NR2C (3) subunit with respect to exon-intron organization, transcriptional start site, alternatively spliced transcripts, and nucleotide differences between gene and transcript and compare the gene organization with that of the NR1 subunit.


MATERIALS AND METHODS

Isolation and Characterization of Genomic Clones

A 129SV murine genomic library in FIX II (Stratagene) was screened for NR2C gene sequences using as probe a 0.9-kb Eco47III rat cDNA fragment (see Fig. 1A). Two overlapping phages, NR2C6 and NR2C4, covering the whole gene were subcloned in pBluescript SK(-) (Stratagene) using SalI of the FIX II polylinker and an internal SalI site to create plasmids pNR2C6 and pNR2C4. The clones were analyzed by dye terminator cycle sequencing (ABI Advanced Biotechnologies, Inc.). The following oligonucleotides were used to prime sequencing reactions: sense: bs11 (5`-AGCTCTGGGCCACTGCAGGC-3`), bs16 (5`-CTCAGCTGCTGGATTTCGTC-3`), rsp18 (5`-GCTGGAGACTGCCGAAGCCACC-3`), bs59 (5`-TGGTTGTGATCGCTCTCAACC-3`), bs6 (5`-CTGACCCTGGCACAGGTGGC-3`), bs15 (5`-TGTAAGGGCTTCTGCATCG-3`), bs26 (5`-GAGACCGGCATCAGTGTGATGGTG-3`), rsp17 (5`-GCTACACGGCCAATCTGGCAGC-3`), rsp15 (5`-CCACTGGCTATGGCATTGCC-3`), rsp16 (5`-GGGGAAGCTGGACGCCTTC-3`), bs2 (5`-GCGGTGGCCAGTGCGAGTCG-3`), bs13 (5`-GAACAGCTGGCTCGGCGGGAG-3`), bs21 (5`-CAGCCACAGTCCCTGGCTC-3`), and bs28 (5`-CAGGGAAGCTTGTGGGACACAAG-3`); and antisense: bs10 (5`-GTGGCTGGATCTCCAGAGGC-3`), bs29 (5`-CAGCCCTGCCCAAGCACCAAGGAG-3`), bs8 (5`-CGGTGCTTCCCAGCGCCAG-3`), bs24 (5`-CTGCTCCAGGGACACGCCCAGCTG-3`), bs25 (5`-GTGCTGTAGCGAGGCCATACTGG-3`), bs18 (5`-AGTCGTAGGAGAACTTGAC-3`), bs14 (5`-ACATCATCACCCACACGGC-3`), bs5 (5`-TCTTGTCACTAAGGCCCGAC-3`), bs20 (5`-GTTCTCGATGGGAACAGAG-3`), bs3 (5`-TGCCATAGCCAGTGGTGGC-3`), bs23 (5`-CATGTTGTCAATGTCCAGC-3`), and bs1 (5`-TATGGGTGTGCAGGCAGAC-3`). Additionally, the following intronic primers were used: sense: bs61 (5`-GCTGGGTCTGAGGATGTGACCAG-3`), bs17 (5`-GTCCTAGTCAGAGCCACATC-3`), bs54 (5`-CCAGTGATGAAGGGTGTGAGG-3`), bs4 (5`-GTGTCTAAGACTGTGCAGG-3`), bs60 (5`-CAGAGAAGCTCTCCGTCTGAG-3`), and rsp14 (5`-GGGAGCACACAGTTAGGAC-3`); and antisense: bs7 (5`-GATAAGGATTCTAAGACCCAG-3`), bs52 (5`-CACCGTCCCACCTGCAGAGGAC-3`), bs55 (5`-CGCATCCCTCTAACATGGGAG-3`), and bs27 (5`-TCCTAGTCGGGAGAAGTCCAGCAG-3`).


Figure 1: The murine NR2C gene and its expression in adult mouse brain. A, schematic drawing of the NR2C gene organization. The exon-intron organization is shown in the upper part. Exons 1-15 are boxed, and shadedboxes represent untranslated exonic areas. Intron 3, which is 6 kb in length, is not drawn to scale. The two alternative splice variants between exons 3 and 4, restriction enzyme cleavage sites (S, SalI; H, HindIII; X, XbaI; K, KpnI), and the positions of the translational start (ATG) and stop (TGA) codons are indicated. The exon alignment of the NR2C cDNA is depicted in the lower part. Coding areas for the four putative transmembrane regions (M1 to M4) are shown by filledboxes. Solidlines below the cDNA and below the genomic DNA indicate the in situ hybridization oligonucleotides for splice variant 1 (a), splice variant 2 (b), the in vitro RNA antisense probe of the RNase protection assay (c), and cDNA probes for the Northern (d) and genomic Southern (e) hybridizations. B, in situ hybridization of horizontal mouse (1, 3) and rat (2, 4) brain sections using specific oligonucleotides for splice variant 1(1, 2) and splice variant 2(3, 4) . C, analysis of the transcriptional start site by RNase protection. Total RNA (50 µg; lane1) from BALB/c mouse cerebellum and Escherichia coli tRNA (50 µg; lane2) were protected with a P-labeled antisense RNA generated by in vitro transcription of a 431-bp genomic fragment derived from the 5`-untranslated region (positions -1142 to -711) and resolved on a 6% polyacrylamide gel. The protected RNA species that correspond to the transcriptional start sites mapped by the RACE protocol and cDNA analysis are indicated by arrows. Approximate RNA sizes are in nucleotides. D, Northern blot analysis depicting the 4.9-kb NR2C mRNA in 10 µg of poly(A) RNA of BALB/c mouse cerebellum. E, Southern blot analysis of 10 µg of mouse liver DNA digested with KpnI (laneK), XbaI (laneX), and HindIII (laneH) revealing fragments of expected sizes (see A) when hybridized with a P-labeled rat cDNA fragment covering exons 12-15.



In Situ Hybridization

In situ hybridization on horizontal sections of adult mouse and rat brains was performed as described (1) using for the large splice variant the selective antisense oligonucleotide bs42 (5`-GCCAGAGTACAGAGAACCTTCCTAGTCCAAGCACA-3`) and for the small splice variant the oligonucleotide bs41 (5`-ACCCATGTCCACTGGAGGGTCCTAGTCCAAGCACA-3`). Hybridization was at 37 °C in 5 times SSC, 40% formamide. Sections were washed in 1 times SSC at 55 °C and exposed to x-ray film at -70 °C for 2 weeks.

Northern Analysis

Poly(A) RNA (10 µg) isolated from mouse cerebellum was resolved on a 1% agarose gel containing 6% formaldehyde. The gel-resolved RNA was transferred onto a nylon filter. The filter was probed with a P-labeled 0.6-kb Eco47III rat cDNA fragment covering exons 9-12 (see Fig. 1A). Hybridization conditions were 5 times Denhardt's solution, 3 times SSC, 0.12 M Na(2)HPO(4), 5 mM EDTA, 1% SDS, 200 µg/ml salmon sperm DNA, 50% formamide at 42 °C for 12 h. The membrane was washed in 2 times SSC, 0.1% SDS at 55 °C and exposed to x-ray film at -70 °C.

Genomic Southern Blot Analysis

BALB/c mouse liver DNA digested with HindIII, KpnI, and XhoI was resolved on a 0.8% agarose gel and blotted onto a nitrocellulose membrane. The membrane was hybridized with the same P-labeled 0.9-kb Eco47III rat cDNA fragment used for screening the genomic library. Hybridization was carried out in 0.12 M Na(2)HPO(4), 5 times SSC, 5 times Denhardt's solution, 0.1 mg/ml yeast tRNA, 30% formamide for 12 h at 42 °C. After hybridization, the membrane was washed in 0.2 times SSC at 65 °C for 30 min and exposed to x-ray film at -70 °C for 3 days.

Localization of the Two Splice Variants

RNA was isolated from the cerebellums of 3-4-week-old BALB/c mice. Total RNA (5 µg) was transcribed into cDNA using 30 nmol of random 10-mer oligonucleotides (Boehringer Mannheim). Of this cDNA, 2 ng were used as template in a polymerase chain reaction. A sense oligonucleotide (bs37, exon 3, 5`-GCTCTAGATCCCCGACGGCTGAGAGGA-3`) and an antisense oligonucleotide (bs38, exon 4, 5`-CCGAATTCTGCCCAAGCACCAAGGAG-3`) served as primers. These oligonucleotides contain XbaI or EcoRI sites to facilitate subcloning and DNA sequencing of PCR products.

Rapid Amplification of 3`-Ends

To determine the 3`-terminal mRNA sequence including the polyadenylation site, a RACE protocol (CLONTECH) was carried out. From 2 µg of cerebellar poly(A) RNA of 3-4-week-old BALB/c mice, first-strand cDNA was primed using an adaptor (dT) (5`-GAGAGAGAGAGACTCGAGTCGACGCGTCGCGATTTTTTTTTTTTTTTTT-3`). The 3`-end of the cDNA was specifically amplified in a PCR in which the same (dT) and a sense oligonucleotide (bs49, exon 15, 5`-GTTGAGTGGGGCCAACTCACC-3`) were used as primers. Blunt-ended products were gel-purified and cloned into M13mp19 replicative form DNA linearized at the HincII site. Twelve independent clones as judged from the different insert lengths were subjected to sequence analysis.

Rapid Amplification of 5`-Ends

For 5`-end analysis, 30 nmol of random 10-mer oligonucleotides were used to prime cDNA synthesis. The RNA/DNA hybrid was hydrolyzed with 6 N NaOH and purified on glass beads (CLONTECH). A poly(A) tail was synthesized at the 3`-end of the cDNA using terminal transferase (Boehringer Mannheim) for 5 min at 37 °C. Polymerase chain reaction was then performed with two primers: an adaptor (dT) (see above) and an antisense oligonucleotide, bs40 (exon 1, 5`-CGCAAAGGCGCGCCTCCCGCTCTC-3`) or bs48 (exon 1, 5`-CCGAATTCGGGGCTGTGTCCCGGCTGCGCAAAGGCGCGC-3`). PCR products (diffuse bands) were gel-purified and inserted into the pGEM-T vector (Promega). Twenty clones with different insert sizes were sequenced for analysis of the 5`-cDNA.

RNase Protection

Total RNA (50 µg) from mouse cerebellum was hybridized with a P-labeled antisense RNA synthesized in vitro from a cloned mouse NR2C gene fragment (positions -1142 to -712). RNase protection was performed according to (2) . The protected RNA was resolved on an 8 M urea, 5% polyacrylamide gel using a DNA sequencing ladder for size determination. An in vitro generated sense RNA (positions -772 to -731) served as a positive control. Hybridization of this RNA to the radiolabeled RNA probe leads to the protection from RNase of a 42-bp RNA duplex. No protected material was seen by hybridizing the RNA probe to tRNA followed by RNase digestion.

Nucleotide Sequences

The sequences for the 15 exons of the mouse NR2C gene have been deposited in the EMBL/GenBank data base under accession numbers L35014 to L35028. The upstream region of the mouse gene can be accessed under accession number L35029.


RESULTS AND DISCUSSION

Recombinant phages carrying the NR2C subunit gene were isolated from a mouse genomic library using a rat cDNA fragment as a hybridization probe. Detailed restriction and sequence analysis of the exonic and most intronic regions in two recombinant overlapping clones indicated that the coding region of the gene is spread over 12,800 bp. The gene is divided into 12 exons (Fig. 1A), and all nucleotide sequences flanking the exons (Fig. 2B) match the consensus splice donor and acceptor sequences. The mouse genomic coding sequence and the reported cDNA sequence for 3 (10) differ by five nucleotide exchanges. The nucleotide insertion at position 2924 and the subsequent deletion at position 3037 might reflect a sequencing error in the mouse cDNA since the rat cDNA (11, 15) and the mouse genomic sequences share the same reading frame. Nucleotide substitutions at positions 2909, 3293 (C to A), and 3407 (C to T) are unique in the genomic sequences. They affect the amino acid composition of the NR2C domains and therefore might reflect allelic variations or site-specific RNA editing (18) in the mouse NR2C locus.


Figure 2: The nucleotide sequences of the transcriptional start site (A), the exon-intron boundaries (B), and the transcriptional stop site (C) of the mouse NR2C gene. Filledcircles in the 5`-untranslated area mark the transcriptional start site (INR) obtained by the RACE protocol. The opencircle indicates the 5`-end of the cloned rat cDNA. Oligonucleotides bs40, bs48, and bs49 used for RACE analysis are indicated by arrows. Blackarrowheads mark the positions of intronic sequences, including the alternative splice acceptor site in exon 4. Sequences that show similarities to nucleotide motifs that interact with transcriptional regulators are boxed (ec, SV40 enhancer core; ELP, embryonal long terminal repeat-binding protein; Sp1, Sp1-binding site; INR, initiator). The translational start site is represented by a bentarrow, the stop codon is indicated by an asterisk, the polyadenylation signal is underlined, and the 3`-terminal nucleotide of the transcript is indicated by an opensquare.



The NR2C gene exists as a single copy in the haploid mouse genome, consistent with the localization of the NR2C gene to human chromosome 7q25(19) . A Southern analysis of murine liver DNA using as a probe a cDNA fragment specific for exons 12-15 revealed a fragment pattern that was consistent with the restriction map generated for isolated NR2C gene fragments (Fig. 1E).

NR2C mRNA in adult BALB/c mouse brain is 4.9 kb in length (Fig. 1D). This transcript terminates 18 nucleotides 3` of the polyadenylation signal AUUAAA, 385 bp after the translational stop (Fig. 2C), as revealed by specific PCR amplification of the 3`-end of the NR2C mRNA. An additional, minor NR2C transcript of 12 kb has been observed in a different mouse strain(10) , but was not detected in BALB/c. The 5`-untranslated leader sequence of the NR2C gene, 772 nucleotides in length, is interrupted by three introns. This became apparent when the 5`-untranslated sequences of cloned rat NR2C cDNAs were compared with the mouse genomic sequence. Gene and cDNA sequences diverge upstream of base -112 and become colinear in three subsequent exons after a long intron of 6 kb. PCR amplification of the 5`-untranslated region revealed that two alternative splice products exist for the mouse NR2C mRNA (Fig. 1A). These differ by 97 nucleotides, and both forms can be readily detected by in situ hybridization in the granule cell layer of adult mouse cerebellum (Fig. 1B). In adult rat brain, at least two additional splice variants exist, one containing an additional 58 nucleotides of exonic sequence located between exons 3 and 4. The 58-bp exon is present in one of the rat NR2C cDNA sequences (15) and in PCR-amplified products of rat brain NR2C cDNA (data not shown), but was not detected in intron 3 of the mouse gene, as judged by hybridization analysis. For the transcriptional start site of the NR2C mRNA, two positions, -772 and -754 nucleotides upstream of the translational start site, were identified by rapid amplification of 5`-ends (see ``Materials and Methods'') using the antisense oligonucleotides that prime 42 and 61 nucleotides downstream of the putative transcriptional start site (Fig. 2A). These initiation sites were confirmed by RNase protection of radiolabeled antisense RNA produced from a genomic fragment (positions -1142 to -712) with 50 µg of total RNA from mouse cerebellum. One prominent RNA fragment corresponding to initiation site -754 was protected from RNase digestion. The signal for initiation site -772 was weak and diffuse and was only visible after longer exposure times. One of the transcriptional start sites (position -772) is localized in the consensus sequence pattern YYAT/AYY, which is described as the transcriptional initiator for polymerase II transcripts in TATA-containing and TATA-less promoters(20) . As in these initiators, NR2C transcription starts at the first A within this element.

The region immediately upstream of the identified transcriptional start sites lacks consensus CAAT or TATA sequence motifs, but is highly GC-rich (72-85%), similar to the high GC content of the neighboring, untranslated exon 1. Sequences corresponding to motifs implicated in binding transcription factor Sp1 (GGGGCGGGGC, position -813)(21) , the embryonal long terminal repeat-binding protein ELP (CAAGGTCAC, position -1022)(22) , and the SV40 enhancer core sequence (CTTTCCAC, position -1157) (23) are located 41, 250, and 385 nucleotides, respectively, upstream of the major transcriptional start sites. The biological importance of these sites needs investigating.

The NR2C gene is the first NR2 subunit gene for which a structure has been determined. When compared with the exon-intron organization of other characterized glutamate receptor subunit genes, e.g. rat GluR-B(26) , Caenorhabditis elegans C-C06E1.4(27) , chick kainate-binding protein(28) , and rat NR1(17) , a close evolutionary origin of NR1 and NR2C subunits can be confirmed. Only between the mouse NR2C gene and the rat NR1 gene were most introns found to interrupt the coding area of the genes in similar or even identical positions (introns 4, 11, and 12 of the NR2C gene and introns 2, 16, 17 of the NR1 gene) (Fig. 3). The NR2C gene contains fewer introns than the gene for NR1 since some of the exons encoding the N-terminal NR2C subunit domain and exons encoding M2 and M3 are fused. Thus, in agreement with the phylogenetic tree(24, 25) and the fact that the NR1 subunit forms functional homomeric channels, the NR1 subunit can be considered to be the oldest member of the NMDA receptor subunit family.


Figure 3: Comparison of the NR2C and NR1 genes. The exon-intron organization of the NR2C and NR1 genes is depicted. Exon regions are boxed and numbered. Shaded boxes represent untranslated exonic areas. The putative four transmembrane regions M1 to M4, alternative splice sites, translational initiation codons (ATG), and stop codons (TGA) are indicated. Identical exon-intron boundaries in the NR1 and NR2C genes are marked with blackdots.




FOOTNOTES

*
This work was supported in part by Grant BCT 364 from the Bundesministerium für Forschung und Technologie and Grant SFB 317/B9 from the Deutsche Forschungsgemeinschaft. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Recipient of a doctoral fellowship from the Fonds der Chemischen Industrie.

To whom correspondence and reprint requests should be addressed. Tel.: 49-6221-566892; Fax: 49-6221-565894; sprengel{at}sun0.urz.uni-heidelberg.de.

(^1)
The abbreviations used are: NMDA, N-methyl D-aspartate; NR1 and NR2, NMDA receptor subunits 1 and 2, respectively; kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction; RACE, rapid amplification of cDNA ends.


ACKNOWLEDGEMENTS

We thank Anne Herb for providing unpublished sequence data on rat NR2C cDNA clones and Jutta Rami for assistance with the manuscript.


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