Human Molecular Genetics, 2002, Vol. 11, No. 3 331-335
© 2002 Oxford University Press
The human intronless melanocortin 4-receptor gene is NMD insensitive
Department of Pediatric Oncology, Hematology and Immunology, University of Heidelberg and University of Heidelberg—EMBL Molecular Medicine Partnership Unit, Im Neuenheimer Feld 150, D-69120 Heidelberg, Germany and 1European Molecular Biology Laboratory and University of Heidelberg—EMBL Molecular Medicine Partnership Unit, Meyerhofstrasse 1, D-69117 Heidelberg, Germany
Received November 5, 2001; Revised and Accepted November 29, 2001.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Nonsense-mediated decay (NMD) is a phylogenetically widely conserved mechanism that contributes to the fidelity of gene expression. NMD inhibits the accumulation of nonsense- or frameshift-mutated mRNA and thus minimizes the synthesis of truncated proteins with potential dominant negative effects. Yeast and higher eukaryotes use somewhat diverse mechanisms to promote NMD and to discriminate between premature and physiological translation termination codons. NMD in yeast involves the binding of specific RNA-binding proteins to cis-acting exonic elements. In contrast, NMD of the intron-containing genes of higher eukaryotes is splicing-dependent. Here, we investigated the NMD sensitivity of nonsense-mutated transcripts of the naturally intronless human melanocortin 4-receptor (MC4-R) gene. Nonsense-mutated variants of MC4-R transcripts are stable and express truncated proteins that are detectable in the lysates of transfected cells. Thus, the naturally intronless MC4-R gene and probably many other intronless genes fail to be monitored by the NMD pathway.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The neural melanocortin 4-receptor (MC4-R) represents a central molecule in a signaling pathway that is implicated in the regulation of food uptake and body weight (1–3) and therefore is of considerable medical interest. MC4-R is a member of the melanocortin receptor family and represents a G-protein coupled seven transmembrane receptor (GPCR) which activates the adenyl cyclase following stimulation by the melanocortin
-melanocyte stimulating hormone (-MSH) (4,5). The interplay between this physiological agonist of MC4-R and its high affinity antagonist termed agouti-related protein (AGRP) is believed to regulate feeding and energy balance in mammals (6,7). Overexpression of AGRP in mice results in hyperphagia, hyperinsulinemia and obesity (8,9), a phenotype that is phenocopied in MC4-R deficient mice (10). In humans, genetic studies related the melanocortin receptors to genetically determined obesity (11,12). Two naturally occurring MC4-R frameshift mutations that generate premature translation termination codons (PTCs) are associated with a dominant form of obesity (2,3). Nonsense-mediated decay (NMD) represents a post-transcriptional surveillance mechanism that eliminates mRNAs with PTCs that result either from nonsense or from frameshift mutations (reviewed in 13–18). The biological and medical importance of NMD is highlighted by its ability to suppress potential dominant negative effects of C-terminally truncated polypeptides. Excellent examples of these include the genes encoding human ß-globin (19,20) and receptor tyrosine kinase (ROR2) (21), and myosin unc54 of Caenorhabditis elegans (22).
In mammalian genes, the exon–exon junctions generated by the splicing process serve as spatial reference points for the discrimination between proper and improper translation termination events. Accordingly, human genes with deleted introns are NMD resistant (23). In intronless yeast genes, the reference point is set by the exonic downstream sequence element (DSE) that has been characterized in the PGK1 gene of Saccharomyces cerevisiae and binds specific, NMD-promoting RNA-binding proteins (15,24–26). It is an interesting question, whether naturally intronless mammalian genes are NMD resistant or whether these genes are able to support NMD by a splicing independent process. This question was recently addressed by an analysis of the NMD competence of PTC-mutated murine heat shock protein (hsp) 70 and human histone H4 mRNAs. The finding of NMD incompetence of these mRNAs supported the view that NMD in higher eukaryotes cannot be activated independently of splicing (27). However, histone and hsp 70 genes occur in multiple copies throughout the genome. Such genes might have evolved surveillance mechanisms that are independent of NMD and that may not operate in intronless single-copy genes. Here, we investigated the NMD sensitivity of PTC-mutated MC4-R mRNAs as an example of intronless single-copy genes. The product of this gene is a central player in the signaling pathway that controls food uptake, and MC4-R mutations are known causes of obesity.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Nonsense-mutated MC4-R transcipts are immune to NMD
In all eukaryotes examined, mRNAs with PTCs are eliminated by NMD. Here, we analyzed the NMD competence of MC4-R transcripts with PTCs in the 5' and in the 3' part of the mRNA, respectively (Fig. 1).
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Construct NS 94 contains a PTC at position 94. In construct del 211, the open reading frame (ORF) is frameshifted by a 4 nt deletion that results in a PTC at position 215, 118 codons 5' of the physiological termination codon. Del 211 is a naturally occurring mutation that leads to severe obesity in the heterozygously affected individuals (3).
The constructs were transiently expressed in HeLa cells together with a wild-type ß-globin construct to control for transfection efficiency. RNAse protection analysis (RPA) of the cytoplasmic RNA revealed that the wild-type (Fig. 1C, lane 3) and both PTC-mutated (Fig. 1C, lanes 4 and 5) MC4-R mRNAs are similarly expressed, and hence that the mutated transcripts do not appear to be subjected to NMD.
NMD incompetence of a nonsense-mutated transcript may result from a lack of translatability (15,28–30). Therefore, it was important to ascertain that the mRNAs that are encoded by the MC4-R constructs used here are translated in the transfected cells.
Both the full-length (Fig. 2, lane 2) MC4-R protein expressed from the 3 x HA-tagged wild-type gene and the truncated (lanes 3 and 4) MC4-R polypeptides expressed from the NS 94 and del 211 constructs are detectable in immunoblots. This shows that the PTC-mutated MC4-R transcripts are translatable. The abundance of the mutated proteins is lower than that of the wild-type MC4-R protein. It has been shown for other GPCRs that the C-terminal part of the receptor is required for efficient transport from the endoplasmic reticulum (ER) to the cell membrane (31). Although this issue is not directly addressed here, the reduced level of mutated MC4-R in these cell lysates is most likely due to ER trapping and subsequent degradation.
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In theory, the lack of NMD may be caused by the presence of a hypothetical sequence element with a stabilizing activity, such as the stabilizing element identified in yeast (15). Such a mechanism has been excluded in multiple copy intronless genes (27).
Therefore, we conclude that the lack of splicing of nonsense-mutated MC4-R transcripts results in NMD incompetence. Furthermore, MC4-R transcripts do not appear to contain cis-acting sequences analogous to the yeast DSE.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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NMD is a surveillance mechanism that contributes to the quality control of gene expression. It monitors mRNAs for the presence of premature termination codons and subsequently eliminates the affected mRNAs (13–18). Nonsense (NS) mutations have been estimated to account for 25% of all mutations that cause genetic diseases (13). The deleterious consequences of the accumulation of truncated proteins expressed from PTC-containing genes has been well documented for ß-thalassemia in humans (19,20) and unc-54 in C.elegans (22). Thus, the suppression of dominant negative effects exerted by incomplete polypeptides represents an important protective role of NMD for heterozygous carriers of mutations. The discrimination mechanism between premature and proper translational termination differs, at least partly, between organisms. In most metazoan genes, this distinction is made by the recognition of the position of a termination codon relative to the last exon–exon junction of the mRNA (30,32–34). Consequently, transcripts expressed from artificially intronless human ß-globin genes are immune to NMD (23). In yeast genes, the proper position of translation termination is defined by its relative position to the DSE. In all organisms, NMD is intimately linked to translation and most models postulate a ribosome-dependent activity that identifies either the relative position of a characteristic assembly of RNA-binding proteins that associate by splicing or the yeast DSE (25,35–38).
Approximately 5% of all human genes are estimated to be naturally intronless. One major class of these genes is represented by the multi-copy histone and hsp genes. These genes have been shown to be immune to NMD (27). Histone and hsp genes are expressed at a very high level. Therefore, the accumulation of nonsense-mutated transcripts and C-terminally truncated polypeptides arising from a single mutant copy of these multi-copy genes may pose relatively little danger of damage. It is possible that potential dominant negative effects may not be as noticeable if only a small proportion of the total number of transcripts encoding a particular product is affected by a truncating mutation. Likewise, a potential gain of function caused by a truncating mutation may not be as noticeable, if only a minor proportion of the transcripts is affected. This may diminish the evolutionary pressure to develop a mechanism of RNA surveillance for this class of genes.
In contrast, cell surface receptor genes and particularly those that encode G-protein coupled receptors represent a class of intronless single-copy genes (39). Because these receptors are central components of important signaling pathways with multiple protein–protein interactions on both sides of the membrane, the expression of truncated variants may be expected to exert dominant negative effects or show a gain of function. Therefore, it was critical to assess the NMD competence of these genes and to analyze whether these naturally intronless single-copy genes have retained or developed a mechanism of RNA surveillance that operates independently of splicing.
MC4-R represents an example of such single-copy GPCRs. Mutations of MC4-R cause dominantly inherited obesity. The naturally occurring PTC mutation del 211 results in a truncated protein lacking the C-terminal 118 amino acids including the 28 amino acids of the cytoplasmic tail. This cytoplasmic tail contains a 14 amino acid conserved motif that is necessary for proper trafficking or protein maturation (1,31,40,41). In both MC4-R truncation mutations studied here this motif is lacking. Therefore, the putative transport defect is likely to prevent transport of a dysfunctional receptor to the cell surface where it could potentially exert dominant negative effects.
It is conceivable that RNA surveillance has not developed in the course of the evolution of the intronless GPCR genes, because potential dominant negative effects or pathological gains of function are prevented at the level of protein processing. Irrespective of the evolution of RNA surveillance, NMD in metazoan cells appears to be splicing-dependent and neither multi-copy nor single-copy intronless genes of higher eukaryotes appear to have evolved a general splicing-independent mechanism to activate NMD. Possible exceptions to this rule should be highly informative and further contribute to the understanding of this medically important pathway.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Constructs
The MC4-R wild-type was generated by PCR amplification of the MC4-R 999 bp ORF with its translational initiation and termination codons and part of the 5'- and 3'-untranslated regions (UTRs) from human genomic DNA (sense primer, 5'-TTTTGGATCCCAGGAGGTTAAATCAATTCAGGG-3'; antisense primer, 5'-TTTTGGATCCGTAAATCCACAGTGCCTACAACC-3'). The gene fragment was gel purified, digested with BamHI and inserted into the polylinker of the pBluescript vector (Stratagene) at the BamHI restriction site. The vector contains a CMV promoter that has been shown not to interfere with the NMD sensitivity of the transcripts (42). For protein detection, a derivative of the pCI-neo vector was used, which contains three tandem copies of the hemagglutinin epitope (3 x HA) starting with an AUG codon 17 bp upstream of the XbaI site of the polylinker (43). The MC4-R ORF was inserted into the XbaI–NotI sites of the pCI-neo polylinker in frame with the 3 x HA-tag.
Constructs NS 94 and del 211 were generated by a C
G site-directed mutagenesis (44) in codon 94 and by deleting four base pairs (CTCT) at codon 211 resulting in a PTC at codon 215, respectively. The identity of all constructs was confirmed by DNA sequencing.
The constructs in the pBluescript vector were used for RNA analysis only, the constructs in the pCI-neo vector were used for RNA analysis and protein detection.
Cell culture and transfection
HeLa cells were grown in Dulbecco’s modified Eagle’s medium under standard conditions. Cells were transiently transfected by calcium phosphate precipitation (45) with 30 µg of the test construct and 20 µg of a wild-type ß-globin gene (30) as a control for transfection efficiency. Cells were washed after 20 h and harvested 24 h later.
RNA analysis
Cytoplasmic RNA was isolated as described by Thermann et al. (30). RNAse protection assays were performed according to standard protocols.
The probe for the MC4-R constructs was prepared by in vitro transcription of a plasmid containing the MC4-R wild-type gene from base 766 to 981. The resulting probe was expected to protect a fragment of 216 nt. The ß-globin probe protected a fragment of 125 nt (23).
Autoradiographic signals were quantitated by imaging in a GS-250 molecular imager (Bio-Rad).
Immunoblotting
For the analysis of cytoplasmic protein, lysates of transfected cells were prepared as described by Thermann et al. (30). Equal amounts of total protein were separated on 13% SDS–polyacrylamide gels. Immunoblotting and immunostaining was performed as described by Thermann et al. (30). The immunoblots were first stained using an anti-HA antibody (EuroGentech) at a 1:1000 dilution, then stripped and restained with an anti-ß-globin antibody (30) to control for transfection efficiency. Protein samples from lysates transfected with a 3 x HA-tagged E2F1 gene served as a positive control for the anti-HA antibody.
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ACKNOWLEDGEMENTS |
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This study was supported financially by the Deutsche Forschungsgemeinschaft and by the Fritz Thyssen Stiftung.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +49 6221 56 2303; Fax: +49 6221 56 4339; Email: andreas_kulozik@med.uni-heidelberg.de
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