Structure and Expression of the Variant Melanin-Concentrating Hormone Genes: Only PMCHL1 Is Transcribed in the Developing Human Brain and Encodes a Putative Protein

Agnès Viale1,*, Anouk Courseaux*, Françoise Presse*, Christine Ortola*, Christophe Breton2,*, Daniel Jordan and Jean-Louis NahonGo,*

*Institut de Pharmacologie Moléculaire et Cellullaire, UPR 411 Centre National de la Recherche Scientifique, Valbonne, France; and
{dagger}Anatomie Pathologique, Faculté de Médecine Laënnec, Lyon, France


    Abstract
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
PMCHL1 and PMCHL2 are two copies of the so-called variant melanin-concentrating hormone (MCH) gene that are located, respectively, on human chromosome 5p14 and 5q13 and that emerged recently during primate evolution. They correspond to a 5'-end truncated version of the MCH gene mapped on chromosome 12q23 and encoding a neuropeptide precursor. The gene organization and regulation of the expression of the variant MCH genes in the human brain are the central issues we investigated. First, the structure and fine chromosomal mapping of the 5p and 5q variant MCH genes were established. These revealed several point mutations and length variations of one CA/TA repeat which allow discrimination between each copy. Using a combination of RACE-PCR, RT-PCR, and sequencing analysis, we provided strong evidence for the expression of the PMCHL1 gene but not the PMCHL2 gene in the human fetal, newborn, and adult brains. Sense, potentially coding, RNAs, as well as noncoding antisense RNAs, were identified and displayed a region-specific expression in the human brain. Strikingly, sense unspliced RNAs of the PMCHL1 gene carried a novel open reading frame and may produce an NLS-containing protein of 8 kDa named VMCH-p8. These transcripts were translated in vitro and in transfected COS cells. Therefore, the PMCHL1 gene provides a unique example of the generation of a gene in the Hominoidae lineage which is specifically transcribed in the developing human brain and has the capacity to be translated into a putative novel protein.


    Introduction
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The genetic events associated with the emergence of new functions during evolution continue to be largely debated. It is well recognized that gene duplication and secondary divergence (Ohno 1970Citation ) or exon shuffling (Gilbert 1978Citation ) are the primary means to generate novel genes. Other mechanisms, such as gene fusion through homologous recombination, illegitimate recombination, or retroposition, may give rise to variants with related functions (when sequences arise from paralogous sequences) or genes with novel functions (when fusion occurs between unrelated sequences).

Several examples of chimeric genes that have evolved according to the gene fusion model are now documented in insects (Long and Langley 1993Citation ) or vertebrates (Chen, Devries, and Cheng 1997a, 1997bCitation ). Recently, we provided evidence for the emergence of a chimeric gene in Primates (Viale et al. 1998Citation ). This gene was named the variant melanin-concentrating hormone (MCH) gene based on partial homology with the authentic MCH gene (Breton et al. 1993Citation ; Breton, Schorpp, and Nahon 1993Citation ). The authentic MCH gene was mapped on human chromosome 12q23 (Viale et al. 1997Citation ) and encodes a cyclic peptide which is a likely leptin target (Qu et al. 1996Citation ; Huang et al. 1999Citation ). MCH has also been established as a major regulator of food intake behavior (Presse et al. 1996; Qu et al. 1996Citation ; Rossi et al. 1997Citation ; Shimada et al. 1998Citation ).

The variant MCH gene arose by duplication from the authentic MCH gene and was found at two loci on chromosome 5, 5p14 and 5q13 (Pedeutour, Szpirer, and Nahon 1994Citation ). We recently revealed that the first event of transposition from the ancestral chromosome 12 to chromosome 5p occurred in the Catarrhini before divergence of the Cercopithecidae and that the last duplication on both arms of chromosome 5 took place in the Hominidea lineage (Viale et al. 1998Citation ). Another striking feature of the variant MCH genes is the expression of at least one form in brain areas of adult humans where the authentic MCH gene is silent (Viale et al. 1998Citation ). Therefore, the MCH gene family provides a unique model with which to investigate the switch in brain-specific expression of genes that diverged during primate evolution.

Sequences of the region surrounding the authentic MCH gene have recently been determined (Viale et al. 1997Citation ). Tissue-specific expression of the human MCH mRNA and processing of the MCH precursor have also been explored (Viale et al. 1997, 1999Citation ). In contrast, few studies are available regarding the genomic organization, tissue-specific expression, and protein characterization of the variant MCH genes (Breton, Schorpp, and Nahon 1993Citation ; Miller, Burmeister, and Thompson 1998Citation ; Miller, Thompson, and Burmeister 1998Citation ; Viale et al. 1998Citation ). Here, we established the structure of the 5'- and 3'-end flanking regions of one variant MCH gene, and we refined the mapping of PMCHL1 and PMCHL2 loci on chromosome 5. Variant MCH gene transcripts were identified and characterized by RT-PCR and RACE-PCR, and the specific distribution of these RNAs was investigated using fetal, newborn, and adult human brain. Finally, we tested whether the longest of the open reading frames (ORFs) found in the putative variant MCH mRNA, referred to previously as ORF1 (Viale et al. 1998Citation ), may be translated in vitro and in a cellular model.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Human Tissues
Different brain samples (hypothalamus, cerebellum, hippocampus, cortex, subicullum, and pallidum) were collected from human fetuses and neonates. All of the procedure was done according to the French legislation of parental consent. Brain structures and one spleen sample of adult donors were provided by the National Neurological Research Specimen Bank (Los Angeles, Calif.). The reference, age, gender, cause of death, collected brain structures, and postmortem delay of every individual are listed in table 1 .


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Table 1 Human Tissue Specimens

 
Cell Line DNAs
DNA from somatic Chinese hamster–human cell hybrids HHW213 and HHW1064 were kindly provided by Drs. B. Wirth and J. Schönling (Institut Human Genetik, Bonn, Germany). HHW1064 contains one human chromosome 5 with a large deletion of the region 5q11.2–13.3 and two smaller deletions on 5p and 5q (Warrington et al. 1991Citation ). HHW213 contains only a human chromosome 5p and some segments from the distal region of 5q. Somatic cell hybrids derived from patients with 5p deletions have kindly been provided by Dr. Overhauser, and characterization of these cells was described elsewhere (Overhauser et al. 1994Citation ).

YAC Contig Analysis
YAC contigs spanning the SMA region (Melki et al. 1994Citation ; Lefebvre et al. 1995Citation ) were generously provided by Drs. J. Melki (Institut Necker, Paris, France) and D. Le Paslier (Centre d'Etude du Polymorphisme Humain, Paris, France). The YAC clones were analyzed in detail by PCR.

Subcloning and Sequencing of Variant MCH Gene Fragments
Six genomic clones, named {lambda}hMCH41 and {lambda}hMCH104 (from a {lambda}EMBL3 library) and {lambda}hMCH1, {lambda}hMCH2, {lambda}hMCH3, and {lambda}hMCH 4 (from two {lambda}GEM11 libraries) had previously been isolated (Breton, Schorpp, and Nahon 1993Citation ). All of these clones carried a common BglII fragment of about 10 kb bearing exon IIv and exon IIIv of the variant gene (Breton, Schorpp, and Nahon 1993Citation ; unpublished data). This fragment was subsequently isolated from {lambda}hMCH41 and subcloned in Bluescript SK vector to generate the phMCH-L37 clone (Viale et al. 1998Citation ). Several overlapping fragments were generated after exon III nuclease digestion (Sambrook, Fritsch, and Maniatis 1989Citation ) of phMCH-L37, and the resulting independent DNAs were sequenced by automated fluorescent DNA sequencing (Applied Biosystems model 373A sequence facilities at the Institut de Pharmacologie Moléculaire et Cellulaire [IPMC]). In some cases, parts of the PMCHL genes were amplified from genomic DNA fragments isolated from hybrid YAC or phage clones through a "genome-walk" method and directly sequenced without further subcloning. Briefly, YAC and phage clones were digested separately with five different restriction enzymes generating blunt-ended termini (DraI, EcoRV, StuI, AluI, and PvuII), and the DNA restriction fragments were ligated to a derived-splinkerette adapter (Devon, Porteous, and Brookes 1995Citation ). The genomic libraries thus obtained were then used as templates in nested PCR reactions with gene-specific primers (Inv21L and Inv21L2; j and k, respectively, in fig. 1 ) and the adapter primers Splink1 (5'-CGA ATC GTA ACC GTT CGT ACG AGA A-3') and Splink2 (5'-TCG TAC GAG AAT CGC TGT CCT CTC C-3'). DNA sequences were analyzed using Bisance service software (Dessen et al. 1990Citation ).



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Fig. 1.—Schematic representation of the variant MCH gene. The sequence was deduced from the phMCH-L37 clone (GenBank accession number AF227988). The position of the region of homology with the authentic MCH gene in the sense orientation is noted as MCHv and included exon IIv and exon IIIv. The remaining part of the truncated PMCHL gene is noted as Inv-MCHv. Black and hatched boxes correspond to CA/TA repeats and an Alu sequence, respectively. The BlgII-EcoRV fragment corresponding to the hMCH-L1 probe and the P2/HMCH2 PCR fragment probe are indicated by double arrows. Locations of primers used for PCR analysis are shown (see table 1 ). Open reading frames (ORFs) of at least 25 amino acids are indicated in both orientations. ORF1 is shown below exon IIv

 
DNA Probes
The human MCH cDNA probe is a fragment of pHMCH2 cDNA after digestion by EcoRI and AccI (Presse et al. 1990Citation ). The HMCHL1 probe is a BglII-EcoRV fragment of the phMCH-L37 cloned in pKS vector (fig. 1 ). A PCR fragment hybridizing to the region of exon IIv and intron Bv of the variant MCH gene was designed using HMCH-P1 and HMCH-2 primers and named P1/HMCH2 probe. These probes were labeled with 32P-deoxy-CTP by the random priming method using commercial kits (Promega).

Southern Blotting
Human genomic DNA from CEPH control families (kindly provided by Dr A. Gal, Institut für Humangenetik, Lübeck, Germany) and cell lines was digested with BglII (or six other endonucleases), electrophoresed onto a 1% agarose slab gel, transferred to a nylon membrane, and hybridized with either the P1/HMCH2 PCR fragment probe, which recognized both the authentic and the variant MCH genes (Viale et al. 1998Citation ), or a specific variant probe named hMCHL1 (see fig. 1 and table 2 ).


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Table 2 Primers and PCR Conditions

 
PCR Amplification of Variant MCH Genes
Nonradioactive Detection
Genomic DNAs of cloned DNAs were amplified through PCR reaction and analyzed as described elsewhere (Viale et al. 1998Citation ). The PCR profiles and the optimum temperature of annealing for each primer are indicated in table 2 .

Radioactive Detection
Ten picomoles of one oligonucleotide primer was end-labeled using T4 polynucleotide kinase (Nahon et al. 1989Citation ) and amplified. Templates were 20 ng of DNA from cell lines. 32P-labeled PCR products were electrophoresed on standard denaturing 6% polyacrylamide DNA sequencing gels and exposed to Bio-max film (Kodak).

Variant MCH Gene Transcript Analysis
RNA Isolation
Whole-cell RNA was extracted by a modification of the acid guanidium thiocyanate phenol chloroform method (Chomczynski and Sacchi 1987Citation ). The final RNA samples were resuspended in DEPC-treated water and kept at -80°C.

Reverse-Transcription PCR Experiments
Total RNA from the different brain areas was reverse-transcribed into single-strand cDNA with the moloney murine leukemia virus reverse transcriptase (GIBCO BRL, France) using either poly-d(T) or variant MCH gene–specific primers (UBI3 for sense transcripts and V1L for antisense transcripts; cf. table 2 Go ). This served as a template for PCR using Taq DNA polymerase (Appligene, France), as previously described (Presse et al. 1992Citation ). The RT-PCR products were detected either by Southern blot analysis with the P1/HMCH2 probe or by nested-PCR reaction with primers corresponding to the authentic MCH gene (HPCR4 and HPCR3) or the variant MCH genes (V1L/P4 and V1L/P7 or V1L/P4 and Inv19L/P7; cf. table 2 and fig. 1 ). To reveal genomic DNA contamination, reverse transcription of RNA was performed in the absence of the enzyme, and PCR was carried out under standard conditions. The level of hybridization of variant MCH and GAPDH mRNA was quantified by densitometric scanning and computerized image analysis (NIH Image, version 1.51). The ratio of variant MCH mRNA/GAPDH mRNA was calculated, and the values of this ratio for each RNA sample were compared with the maximal value of the ratio of the control group (hypothalamic RNA sample corresponding to newborn 2) using a four-point scale (-, +, ++, and +++).



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Fig. 6.—Nucleic acid and deduced protein sequence comparison of the MCH and PMCHL1 genes. DNA sequences and deduced amino acid (AA) sequences of exon II of the MCH gene (Breton, Schorpp, and Nahon 1993Citation ) and of the corresponding region of the PMCHL1 gene (fig. 4 ). Differences are boxed in dark (nucleic acid) and gray (protein). The region of homology corresponding to NEI (AA131–AA144 of pro-MCH and AA52–AA65 of ORF1) is boxed. The ORF1 sequence starts at the first methionine (M, boxed at position 1). (*) Stop codon; () intron of the MCH gene

 
Rapid Amplification of cDNA Ends (RACE) Analysis
RACE analysis was done using a Marathon-ready cDNA human fetal brain library (Clontech, Palo Alto, Calif.) according to the procedure of the manufacturer, with primers specific to the PMCHL genes (namely, P1, P4, P4As, P6As, 25, and hPCR3; cf. table 2 Go ). RACE-PCR products were purified and directly sequenced as described above.



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Fig. 5.—Variant MCH gene expression in the human brain. A, RACE-PCR analysis. The 5' end extended products were amplified from a fetal human brain cDNA library using the 5' adapter AP1 and HPCR3 primer. Sequences were obtained with primers specific to the variant MCH gene (P1, P4, P7, ...). The location of ORF1 and the putative exons of the variant MCH gene are indicated. B, RT-PCR analysis of polyadenylated RNAs. Cb = cerebellum ; Hip = hippocampus of adult individuals (see table 1 ). As a control, GAPDH PCR products (0.52 kb) were identified on the ethidium bromide–stained gels. RT = reverse-transcribed; NRT = non–reverse-transcribed. C, and D, RT-PCR analysis of sense and antisense variant MCH RNAs, respectively; cerebellum (Cb), cortex (Cx), and hypothalamus (Hpt) of fetus 3 and newborn 2 (table 1 ). In B and C, the corresponding Southern blots were probed with a 32P-labeled P1/HMCH2 fragment

 
In Vitro Translation Assay
In vitro translation of ORF1 was carried out using the TNT-T7 coupled reticulocyte lysate system (Promega, Wisconsin). A BamH1/EcoR1 fragment encompassing ORF1 (see fig. 7A ) was subcloned into pKS (Stratagene, California), pGEM (Promega), or an IPMC-made expression vector, pEXO, derived from pSK and containing Xenopus ß-globin mRNA sequences which allowed mRNA stabilization (Lingueglia et al. 1993Citation ). Briefly, 1–5 µg of circular recombinant ORF1 DNA was added to 12.5 µl of TNT Rabbit reticulocyte lysate, 1 µl of TNT Reaction buffer, 0.5 µl of T7 TNT RNA polymerase (0.5 µg/µl), 0.5 µl of 35S-methionine (1,000 Ci/mmol, 10 µCi/µl), 0.5 µl RNasin ribonuclease inhibitor (40 U/µl), and 3 µl nuclease-free H2O, for a final volume of 25 µl. The mix was incubated at 30°C for 1.5–2.5 h. To reduce the background generated by the 35S-methionine/tRNA complex, the samples were treated with RNAse A (50 µg/ml) for 10 min at 30°C in the presence of 5 mM EDTA. Translation products were separated onto 15% SDS-page gel. After drying, gels were exposed and analyzed with a phosphoimager (Fuji film BAS 1500) or visualized by autoradiography. As a positive control, luciferase DNA encoding a 61-kDa protein (Promega) was tested. As a negative control, a transcription/translation experiment was performed in the absence of a DNA template.



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Fig. 7.—Expression of VMCH-p8 in vitro and in transfected cells. A, BamH1-EcoR1 fragment coding for ORF1 used for cloning in expression vectors. B In vitro expression of the pEXO-VAR construct. Different amounts of a recombinant pEXO-VAR plasmid (0–1.5 µg) were added to the coupled transcription/translation assay, and proteins were analyzed on a 16.5% SDS-PAGE. Two independent experiments are shown. The position of VMCH-p8 is indicated. C, Expression in transfected COS7 cells. COS7 cells were transfected for 48 h with pCMV-FLAG/ORF1 under two slightly different protocols. Cells were fixed, permeabilized, and incubated with an M2 antiserum (upper panel) or a NEI antiserum (bottom panel), and the primary antibodies were detected with a rhodamine-labeled secondary antiserum (upper panel) or FITC-labeled secondary antiserum (bottom panel). Magnification: x100

 
Transfection of COS Cells with Tagged-ORF1 pCMV Vectors
Plasmid phMCH-L37 was used as a template for the preparation of a PCR fragment containing ORF1, as well as part of the 5'-flanking noncoding regionGo . The resulting DNA was subcloned into the HindIII/ClaI sites of a modified pCMV/FLAG vector (Invitrogen, California). Therefore, the ORF1 was fused in its C-terminus to the FLAG sequence (MDYKDDDDKV). COS-7 or COS-M6 cells, grown to 30%–50% (experiment B) or 75%–80% (experiment A) confluency in 35-mm (Western blot analysis) or 100-mm (immunofluorescence microscopy analysis) tissue culture dishes, were transfected with 1 µg (35-mm plate) or 7 µg (100-mm plate) of the tested plasmid by a modified DEAE-dextran method (Gonzalez and Joly 1995Citation ). After three hours of treatment, the transfection mix (10 µl DEAE-dextran 100 µg/µl; 2 µl chloroquine 60 µg/µl; 2.5 ml DMEM medium/plate) was replaced (experiment A), or not (experiment B), by 10% DMSO for 2 min. In a typical experiment, cells were transfected in duplicate with either pCMV-FLAG/ORF1 (pCF8 plasmid), pCMV-FLAG only (negative control), pCMV-FLAG/IRK3 encoding an inward rectifier K+ channel (Lesage et al. 1994Citation ) or pCMV-GFP (transfection positive control). pCMV-FLAG/IRK3 vector was taken as a control for our transfection/expression conditions in COS-7 cells, and strong staining with the M2 antiserum was found in the cytoplasm and in the membranes, as expected (not shown). The expression of ORF1-containing RNAs was checked by RT-PCR with HMCH45/HMCHR46 primers Go in nontransfected and pCF8-transfected COS cells.

For immunofluorescence microscopy, the transfected COS cells were grown on glass coverslips for 24 h. They were fixed for 15 min with 4% (v/v) paraformaldehyde in 1 x PBS. After rinsing twice with 1 x PBS, cells were permeabilized by incubation for 10 min with 0.1% Triton X-100/2% BSA/1/20 normal goat serum (vector 51000). The anti-FLAG M2 antibody (Kodak, SIGMA; dil. 1/400) and the anti-NEI antibody (dil. 1/500) (Bittencourt et al. 1992Citation ) were incubated with cells for 1 h at room temperature. After three washings with 1 x PBS, fixed cells were incubated with either fluorescein isothiocyanate (FITC)–labeled goat anti-serum anti-rabbit antibody (to detect NEI) or rhodamine-labeled goat anti-mouse antibody (to detect FLAG/M2 epitopes) at 1:200 dilution. The cells were rinsed three times in 1 x PBS and twice with 10 mM Tris-HCl [pH 7.4] buffer and mounted with 2–3 µl of Vectra shield medicine (Vector Lab.) on a glass slide and finally observed under fluorescence.


    Results
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
Flanking-Region Analysis of One Variant MCH Gene
A 4.2 kb-region of the phMCH-L37 clone, containing the entire region of homology with the structural part of the authentic MCH gene, about 2.5 kb of 5'-flanking sequences, and 1.0 kb of 3'-flanking sequences, was entirely sequenced using a mixture of direct sequencing of double-stranded DNA using specific primers, sequencing of deleted fragments subcloned in pSK vector, and sequencing of YAC or phage clones through a genome-walk method. Figure 1 illustrates the organization of the putative regulatory and structural regions of the variant MCH gene found in the phMCH-L37 clone (GenBank accession number Bank It 316642/AF227988). The sequence homologous to the exon II–intron B–exon III region of the authentic MCH gene was previously named exon IIv–intron Bv–exon IIIv (Breton, Schorpp, and Nahon 1993Citation ) and is named MCHv here. A systematic analysis of ORFs reveals one particular sequence of 72 amino acids, previously named ORF1 (Viale et al. 1998Citation ), which overlaps the exon IIv sequence in the same frame as that of the pro-MCH. ORF1 displays a strong sequence identity with the corresponding part of the authentic MCH gene (Breton, Schorpp, and Nahon 1993Citation ). None of the other ORFs displayed sequence homology with known proteins.

Two CA/TA repeats were found in the 5'-flanking region. Analysis of the 3'-flanking sequences revealed the insertion of an Alu-Sq element into a sequence 80% identical to part of the intron A–exon II junction of the authentic MCH gene (fig. 1 ). This sequence, named Inv-MCHv, is oriented in the opposite direction of the MCHv sequence of the variant MCH gene and likely represents the remains of truncation/insertion events of the MCH gene that occurred during primate evolution (Viale et al. 1998Citation ).

High-Resolution Mapping of the Two Variant MCH Genes
We first tried to detect a restriction fragment length difference after cleavage with six different restriction enzymes (including BglII; see fig. 2A ) that may distinguish copies of the variant MCH gene on 5p and 5q. We failed to reveal such a variation by Southern blot analysis using either a panel of 20 human genomic DNAs (5 are illustrated in fig. 2A ) or the six genomic MCH clones derived from different phage libraries (see Materials and Methods; data not shown).



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Fig. 2.—Structural differences between PMCHL1 and PMCHL2 genes. A, Southern blot analysis of human DNA from reference CEPH families. A blot containing BlglII-digested genomic DNAs was sequentially hybridized with the P1/HMCH2 probe (recognizing the authentic and variant MCH genes), then with the hMCH-L1 probe (specific to the variant MCH genes). B, Autoradiogram of a gel after electrophoretic separation of PCR products generated with 32P-labeled HV-GM1/unlabeled HV-GM2 primers. L37 = phMCH-L37; 751E3 = YAC clone 751E3; C = negative control (no genomic DNA); G = human genomic DNA. Bands at 240 and 270 bp were observed in the YAC 751E3 and phMCH-L37 clones, respectively. One band in addition to the 270-bp fragment was visualized in lane G because the selected individual was a heterozygote for the corresponding locus. C, Mapping of the PMCHL1 gene using a panel of hamster/human cell hybrids carrying deletions of human chromosome 5p13–15. The figure shows a pattern of 32P-labeled PCR products amplified from DNA of representative hybrid cell lines (see fig. 3 ).

 
However, by using primers HV-GM1 and HV-GM2 (a and a', respectively, in fig. 1 ), flanking one of the newly identified CA/TA repeats, we were able to distinguish the two variant MCH genes, PMCHL1 and PMCHL2. PCR amplification generated a 240-bp product for PMCHL2 versus a 270-bp product for PMCHL1 (fig. 2B ). The 270-bp product was associated with the PMCHL1 locus, since it was present in genomic DNA from HHW1064 and HHW213 cell lines carrying deletions in the 5q13 region.

To determine the precise location of the PMCHL1 gene on 5p14, a PCR screening strategy was used. DNAs isolated from somatic cell hybrids containing deletions of human chromosome 5p (Overhauser et al. 1994Citation ; fig. 3 , top) were amplified with 32P-labeled HV-GM1/unlabeled HV-GM2 primers which detected both PMCHL1 and PMCHL2. A representative example of the hybridization pattern obtained is shown in figure 2C. As expected, the 240-bp PCR product was found in every DNA sample. In contrast, the 270-bp PCR product was present or absent, depending on the length of the deleted region of chromosome 5. Based on these data, the PMCHL1 locus was mapped at the distal end of 5p14.3 (fig. 3 , top).



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Fig. 3.—Mapping of PMCHL1 and PMCHL2 genes on human chromosome 5. Upper part, Mapping of PMCHL1 on chromosome 5p. The presence (+) or absence (-) of specific PCR products corresponding to the PMCHL1 locus is indicated for each hybrid cell line noted above. The black bar indicates the extent of the nondeleted region on 5p (Overhauser et al. 1994Citation ). The gray box indicates the region of the PMCHL1 locus. Lower part, Mapping of PMCHL2 on chromosome 5q. YACs overlapping and flanking the SMA locus region are shown. The location of the variant MCH gene (PMCHL2) is indicated both by an arrow in 5q13.3 and on the YAC clones 798B3, 742E10, and 751E3. Chromosomal regions involved in previously described clinical phenotypes are indicated on the left by a dark line

 
To elucidate the precise location of the PMCHL2 locus on 5q13, we used a series of YAC clones spanning the SMA locus (Melki et al. 1994Citation ; Lefebvre et al. 1995Citation ). A specific PMCHL2 PCR product, amplified with the P1/2B primers, was identified only with YACs proximal to the SMA locus (namely, 751E3, 742E10, and 798B3). Therefore, the PMCL2 locus was mapped on chromosome 5q13.3 (fig. 3 , bottom).

Sequence Analysis Revealed Several Point Mutations Between PMCHL1 and PMCHL2 Genes
Inasmuch as different CA/TA repeat lengths could be selectively associated with one of the two variant MCH genes, it was now possible to ascribe the variant MCH gene sequences to either of the chromosome 5 loci. Since the 270-bp PCR product was identified on all our phage clones and on phMCH-L37 (fig. 2B, lane L37), those clones encompassed the PMCHL1 gene.

To characterize PMCHL2, different PCR products were generated from either YAC clones (742E10 and 751E3; fig. 3 ) or cell hybrids carrying deleted regions of chromosome 5p (JH132 and HHW740; fig. 3 ) using primers that bracketed the putative coding part of the variant MCH gene. The sequences were identical among these DNAs but exhibited several reliable differences when compared with the phMCH-L37. As shown in figure 4 , 22 mutations were distributed along the sequence between HMCH379 and HMCH7 primers and f', respectively, in fig. 1 ). Some of them were associated with restriction cleavage sites, such as the MaeI, AvaII, and PvuII sites at positions 2664, 2828, and 3010, respectively, that allowed discrimination between the two variant MCH genes (GenBank accession numbers: PMCHL1, AF238382; PMCHL2, AF238383).



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Fig. 4.—DNA sequence comparison of the PMCHL1 and PMCHL2 genes. PCR fragments were generated from YAC clones (742E10 and 751E3) or cellular hybrids (JH132, HHW740) by different sets of primers to amplify the region of exon IIv–intron Bv–exon IIIv of the PMCHL2 gene. The consensus sequence was compared with that of the PMCHL1 gene established from the phMCH-L37 clone. Putative exon/intron regions are shown. Mutations/deletions are represented by boxes. * MaeI (5q); ** AvaII (5q); *** PvuII (5p). GenBank accession numbers: PMCHL1, AF238382; PMCHL2, AF38383

 
Analysis of Transcripts from the Variant MCH Genes
To identify and establish the structure of RNAs generated from the variant MCH genes, a RACE approach was taken first. A specific primer recognizing the putative coding region of the variant MCH gene (HPCR3; fig. 5A ) was used with adapter AP1 and AP2 primers to generate fragments elongated in the 5' terminus of transcripts present in a human fetal brain cDNA library. A RACE product of 800 bp in the 5' end direction was consistently found and further characterized from this "marathon" RACE cDNA library. This 5' RACE-PCR product corresponded to an unspliced RNA that overlapped the MCHv region and contained ORF1.

Thereafter, reverse transcription and PCR amplification were used to reveal the expression of variant MCH gene transcripts in different areas of fetal, newborn, and adult human brains (table 3 ). Polyadenylated forms of the variant MCH RNAs were first analyzed using oligo d(T) for reverse transcription and HMCH-P1 and HMCH2 primers (see table 2 ) for PCR amplification. A single product of 570 bp was identified in different RNA samples extracted from hippocampus and cerebellum of adults (fig. 5B ). Direct sequencing of this fragment showed that it was colinear with the genomic sequence of PMCHL1, indicating that it corresponded to unspliced transcripts of that gene (not shown). Contamination with genomic DNA appeared unlikely due to the absence of amplification in the non–reverse-transcribed (NRT) samples using specific-variant MCH gene primers or specific-GAPDH gene primers (fig. 5B ). The 570-bp PCR product was found in the hippocampus (or cortex), cerebellum, and hypothalamus at all stages of human development that we analyzed (table 3 ). In contrast, this product was lacking in the cerebellum and spleen of adult 2348 (fig. 5B ), as well as in all peripheral fetus and newborn tissues (not shown).


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Table 3 Expression of Polyadenylated, Sense, and Antisense Variant MCH RNAs in Different Brain Areas During Human Development

 
Specific primers were employed during reverse transcription to reveal sense (UBI3 primer) or antisense (V1L primer) variant MCH RNAs (see open arrows in fig. 5A for locations and orientations of the primers). A representative Southern blot of PCR DNA fragments produced with V1L and HMCH-P4 primers after reverse transcription with UBI3 is shown in figure 5C. Since the first PCR products generated with V1L/P4 primers were not abundant enough to allow direct sequencing, a second nested PCR amplification was performed by using a primer upstream from P4 (primer P7; see fig. 5A ). An ethidium bromide–stained gel illustrated RT-PCR product analysis of fetal RNA after reverse transcription with the V1L primer and nested PCR (fig. 5D ). A unique fragment of 0.42 kb was found in the regions of the brain where both sense (fig. 5C ) and antisense (fig. 5D ) variant MCH gene transcripts were identified. Direct sequencing of these fragments showed that they represented unspliced RNAs transcribed exclusively from the PMCHL1 gene, in agreement with our sequence analysis of the polyadenylated variant MCH transcripts.

As summarized in table 3 , sense variant MCH RNAs were found in at least one cortex and cerebellum at all stages of human brain development, whereas these transcripts were absent in the hypothalami of two fetuses, one neonate (our present data), and all adults we tested previously (Viale et al. 1998Citation ). The antisense variant MCH RNAs were clearly identified in the cortex, hippocampus, and hypothalamus at all stages of development we analyzed. However, these antisense RNAs were not strongly expressed in cerebellar extracts from fetuses and adults.

Expression of a Putative Translational Product from the Variant MCH Gene In Vitro and in Transfected COS Cells
The longest ORF deduced from the RACE-PCR and RT-PCR DNA sequences corresponded to ORF1. It initiated from an ATG codon (methionine, boxed in fig. 6 ) in a reasonable Kozak context (Kozak 1991Citation ) and had a purine A at the -3 position. This sequence potentially encodes a short protein of 72 amino acids (8 kDa), designated here variant (V)-MCH-protein (p)8 (VMCH-p8). The VMCH-p8 sequence exhibits strong homology (89% identity at the protein level) to the part of pro-MCH encoded in exon II of the MCH gene (fig. 6 ). Sequence conservation was marked in the region of the NEI peptide which was located upstream of the MCH sequence in the same precursor of rats, mice, and humans (Nahon et al. 1989Citation ; Presse et al. 1990Citation ) (amino acids 131–144 of pro-MCH, amino acids 52–65 of VMCH-p8; boxed in fig. 6 ). Several nucleotide substitutions were noted, but many of these changes resulted in amino acid conservations (Asn90, Phe114, Gly119, Thre123). Sequence differences were confined to the N-terminal part (amino acids 1–8 of VMCH-p8) and the C-terminus (amino acids 71–72 of VMCH-p8).

Predicted hydrophobicity and secondary structure plots of VMCH-p8 (not shown) indicated that this protein lacked an initial stretch of hydrophobic residues that could act as a signal peptide. Therefore, VMCH-p8 is unlikely to be a secreted protein or a neuropeptide precursor. Sequence analysis using PSORT II programs predicted that VMCH-p8 could be a nuclear protein (reliability 52.2% with the k-NN prediction test). A stretch of basic residues is located at amino acids 5–9 of VMCH-p8 (underlined in fig. 6 ). This sequence corresponds to a classical nuclear localization signal (NLS) according to the PSORT II subprogram.

To assess the translational potential of the RNA encoding VMCH-p8, a PCR fragment encompassing ORF1 was produced and introduced at the BamH1-EcoR1 sites in pKS or pGEM vectors (see fig. 7A ). These constructs were added to a coupled transcription/translation system (TNT-T7 coupled reticulocyte lysate kit). We failed to reveal a variant MCH protein using these constructs. Using the pEXO-VAR construct, which carries ß-globin mRNA-stabilizing sequences, a single protein of the expected molecular mass was identified, its synthesis depending on the amount of pEXO-VAR construct added to the TNT-T7 translation system (fig. 7B ).

A pCMV-FLAG/ORF1 construct was transfected to COS-7 cells under two different conditions of transfection (experiments A and B, Materials and Methods), and indirect immunofluorescence microscopy was used with either anti-FLAG M2 antiserum or anti-NEI antiserum. A similar punctate pattern of fluorescence was observed with the M2 antiserum (fig. 7C, upper panel) and the anti-NEI antiserum (fig. 7C, bottom panel). The majority of the tagged protein accumulated in the perinuclear space of the endoplasmic reticulum (arrowhead in fig. 7C ), while a small fraction was associated with the nucleus (arrow in fig. 7C, bottom panel). Cells transfected with the pCMV vector alone displayed only fluorescent background (not shown).


    Discussion
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
The evolutionary mechanisms that led to the emergence of the variant MCH gene in the Primates lineage, as well as partial structure of the gene, were previously established (Viale et al. 1998Citation ). This variant gene arose by duplication/truncation from an ancestral MCH gene about 30 MYA between the divergence of Platyrrhini and Catarrhini apes. Then, another duplication in the Hominidae group, about 10 MYA, gave the pattern observed in modern humans. This situation offers an unprecedented opportunity to analyze the molecular mechanisms of gene remodeling and selection of functions which operate during primate evolution.

First, we extended the sequence analysis of the human variant MCH gene to the 3' and 5' regions flanking the exon-intron structure previously described (Breton et al. 1993Citation ). Surprisingly, a sequence corresponding to the intron A–exon II part of the authentic MCH gene was found located in an inverse orientation at the 3' end, close to the MCHv region of the variant MCH gene. An Alu-Sq sequence was also identified inserted into the so-called Inv-MCHv region bracketed by short direct repeats, as expected at a site of mobile element insertion. Because the retrotransposition of the Alu-Sq subfamily occurred about 44 MYA (Mighell, Markham, and Robinson 1997Citation ), this Alu repeat could be involved in the truncation process that led to the creation of the variant MCH gene in the equivalent of chromosome 5p14 in the Catarrhini ancestor (Viale et al. 1998Citation ). Alternatively, it was recently transposed during primate evolution. Sequencing of the 3'-flanking region of the variant MCH gene in Cercopithecinae species should clarify this issue.

Two CA/TA repeat elements were mapped close to the exon IIv–intron B–exon IIIv region of the variant MCH gene. One of these repeats displayed different lengths, depending on whether it came from the 5p or the 5q copy of the variant MCH gene. We established therefore that the phMCH-L37 carried the sequence of the 5p locus and that multiple point mutations clearly differentiate the PMCHL1 and PMCHL2 genes (22 positions in a 1.1-kb fragment encompassing the exon IIv–intron Bv–exon IIv region). These results confirmed and extended previous data obtained from PCR-SSCP to a radiation hybrid panel (Miller, Thompson, and Burmeister 1998Citation ). In particular, the site for the PvuII restriction enzyme is lacking in the PMCHL1 sequence, and it may be conveniently used to discriminate the two variant MCH genes and transcripts. We used this technique extensively to characterize the transcripts revealed in the human brain (see below).

Using the size difference of the CA/TA repeat, we precisely located the PMCHL1 and PMCHL2 genes on human chromosome 5, refining the map proposed by Miller, Thompson, and Burmeister (1998)Citation . PMCHL1 was assigned to a 1-Mb region in chromosome 5p14.3. Deletions covering this region do not contribute to the cri-du-chat syndrome or mental retardation (Overhauser et al. 1994Citation ). However, PMCHL1 may be a candidate gene for multiple sclerosis (Ebers et al. 1996Citation ; Kuokkanen et al. 1996Citation ) and craniometaphyseal dyplasia (Nurnberg et al. 1997Citation ). On chromosome 5q13.3, the variant PMCHL2 gene was found in YAC clones 798B3, 742E10, and 751E3, close to the SMA locus. However, the PMCHL2 gene does not lie within the critical region where deletions, gene conversions, or mutations have been associated with clinical expression of the disease (Melki et al. 1994Citation ; Lefebvre et al. 1995Citation ; Roy et al. 1995Citation ). Thus, it is unlikely that PMCHL2 may be directly involved in the SMA phenotype. However, PMCHL2 falls in a region of susceptibility for the Wagner disease and erosive vitreoretinopathy (Brown et al. 1995Citation ) (see fig. 3 ). Nevertheless, these results do not provide direct evidence that either of the variant MCH genes is associated with these diseases. In this context, a broad region of chromosome 5 was linked to obesity in French families (Hager et al. 1998Citation ), but its association with PMCHL1 and PMCHL2 loci is largely hindered by the inability to pinpoint the locus.

An important aspect of the work presented here is the demonstration that both sense and antisense transcripts from the PMCHL1 gene are present in different areas of the developing human brain. We also demonstrate that PMCHL2 is totally silent during development of the human brain. The divergent expression patterns of PMCHL1 and PMCHL2 are likely the result of a different genomic environment in the flanking region, but this remains to be established.

Miller, Burmeister, and Thompson (1998)Citation have previously identified antisense unspliced RNAs from PMCHL1 in the hypothalamus of one adult human. We confirmed these data and showed also that antisense RNAs overlapping the MCHv region were widely produced in fetal, newborn, and adult brains. No ORF of significant length existed in the antisense orientation of the MCHv region (fig. 1 ). This raises the possibility that antisense PMCHL1 transcripts function as untranslated RNAs. Direct interaction with the complementary sense transcripts originating from either the variant MCH gene or the authentic MCH gene may contribute to control splicing, stability, and/or translational efficiency (reviewed by Dolnick 1997Citation ; Vanhée-Brossollet and Vaquero 1998Citation ). Considering the expression patterns of the sense and antisense PMCHL1 gene transcripts in the human brain, they fulfill the criteria for the "determinator-inhibitor pair" model proposed by McCarrey and Riggs (1986)Citation . Alternatively, there are a growing number of examples of RNAs lacking extensive ORFs which regulate important functions without direct interaction with complementary transcripts (reviewed in Erdmann et al. 1999). Some of these untranslated RNAs play a role in gene regulation during normal development, such as those transcribed from the Xist gene, involved in the X-chromosome inactivation process (Brown et al. 1992Citation ). Others act as tumor suppressors and modulate cell growth, such as the noncoding RNAs synthesized from the H19 gene (Brannan et al. 1990Citation ; Hao et al. 1993Citation ).

Sense unspliced transcripts from PMCHL1 were identified at all stages of human development in the cortex and cerebellum but not in the fetal and adult hypothalami (see table 3 ; Viale et al. 1998Citation ). In addition, no expression of these sense RNAs was found in one fetus and one newborn. Although the basis for this difference is unknown, it may be related to the postmortem interval (PMI) and the agonal state (events prior to death) (Barton et al. 1993Citation ). Indeed, the lack of expression of the sense transcripts correlated with the longest PMI in fetal tissues (see tables 1 and 3 ). In addition, hypoxia during the premorterm period in SIDS neonates may also influence the stability of mRNA as found in animal models (White and Lawson 1997Citation ). Thus, quantitative assessment of PMCHL1 gene expression during human brain development is difficult to evaluate. In a similar respect, unspliced sense transcripts are absent in the hypothalamus at all stages of development, except for one newborn (case 2). Again, the proximal cause of death may have selectively influenced PMCHL1 gene expression in this particular case.

The sense PMCHL1 gene transcripts identified in the human brain contain a coding sequence (ORF1) of 72 amino acids which corresponds to an 8-kDa protein named VMCH-p8. Because of the lack of a signal peptide (originally present in exon I of the MCH gene), this ORF has probably lost its original function. In addition, the absence in the human brain of PMCHL1 transcripts where intron Bv is spliced indicates that the production of a putative variant MCH peptide is unlikely. Indeed, by using RP-HPLC coupled with a specific RIA, we failed to identify this variant MCH peptide in various brain regions of fetus, newborn, or adult individuals (unpublished data). However, an intriguing feature of the exon IIv sequence is the high degree of conservation between the MCH and PMCHL1 genes at the DNA and protein sequences both in humans (fig. 6 ; Breton et al. 1993Citation ) and in the variant orthologs in Primates (Viale et al. 1998Citation ). This likely reflects a constraint in the ORF1 sequence. Arguments for the translational capacity of the ORF1-containing RNAs come from in vitro translation and COS cell transfection experiments shown here. An NLS motif was identified in the N-terminal part of VMCH-p8 (see fig. 6 ) which could contribute to directing this protein to the nucleus, as found for other nuclear proteins (Nigg 1997Citation ). This hypothesis is currently being tested with constructs deleted or mutated in the putative NLS sequence and transfected into different cell types.

In conclusion, the variant PMCHL1 gene represents a unique example of the assembly of unrelated sequences through a complex rearrangment process that conveyed the conversion of a neuropeptide-encoding gene (i.e., a secreted protein) to a gene encoding a putative nucleus-targeted protein (i.e., an intracellular protein) during primate evolution. All of the requirements for PMCHL1 being a new expressed gene, or a gene "in search of a function" (Marshall, Raff, and Raff 1994Citation ), are present here. It is transcriptionally active, albeit at a low level. This means that insertion of the transposed ancestor of this gene occurred in a favorable environment of cis-regulatory elements driving the striking tissue-specific expression of the variant PMCHL1 gene. This is at odds with the other copy of the variant MCH gene (PMCHL2), which appeared not to be transcribed at all in the human brain. Most interestingly, the active PMCHL1 gene may code for a protein, VMCH-p8, but proof of expression of this protein in vivo is presently lacking. The next challenge is to characterize VMCH-p8, as well as other putative variant MCH proteins in developing human brain, and to elucidate their function(s).


    Acknowledgements
 TOP
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Acknowledgements
 literature cited
 
We give many thanks to Drs. B. Saatkamp, P. Vernier, and P. Kitabgi for helpful advice and critical reading of the manuscript. Brain samples from fetuses and newborns were kindly provided by Drs. R. Bouvier and J. Dutruge (Faculté de médecine A. Carrel, Lyon, France). The adult brain structures were generously offered by the National Neurological Research Specimen Bank (Los Angeles, Calif.), which is sponsored by NINDS/NIMH, the National Multiple Sclerosis Society, the Hereditary Disease Foundation, and the Veterans Health Services and Research Administration, Department of Veterans Affairs. We thank Dr. J. Overhauser (T. Jefferson Medical College, Philadelphia, Pa.) for providing cell hybrid DNAs. The anti-NEI antiserum was kindly provided by Dr. J. Vaughan and Prof. W. Vale (the Salk Institute, La Jolla, Calif.). This work was supported by the Groupement de Recherches et d'Etudes sur les Génomes (GREG 100/94) and by the Association Française contre les Myopathies (AFM) (ASI 1996–1998). A.V. was a recipient of fellowships from the ADER-PACA (CAR 9312/2679; 1994–1996) and the AFM (1997). A.C. was supported by the AFM (1996–1997) and the Association pour la Recherche sur le Cancer (1997–1999).


    Footnotes
 
Claudia Kappen, Reviewing Editor

1 Present address: Laboratory of Molecular Genetics, Rockefeller University, New York. Back

2 Present address: Centre National de la Recherche Scientifique INSERM de Pharmacologie-Endocrinologie, Montpellier, France. Back

3 Keywords: variant MCH gene antisense RNA human brain human chromosome 5 expressed pseudogene primate evolution Back

4 Address for correspondence and reprints: NAHON Jean-Louis, Institut de Pharmacologie Moléculaire et Cellulaire, Centre National de la Recherche Scientifique UPR 411, Sophia-Antipolis, 06560 Valbonne, France. E-mail: nahonjl{at}ipmc.cnrs.fr Back


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Accepted for publication July 13, 2000.