The 350-fold compacted Fugu parkin gene is structurally and functionally similar to human Parkin

Gene 346 (2005) 97 – 104 www.elsevier.com/locate/gene

The 350-fold compacted Fugu parkin gene is structurally and functionally similar to human Parkin Wei-Ping Yua,b, Jeanne M.M. Tanc, Katherine C.M. Chewc,d, Tania Ohe, Prasanna Kolatkare, Byrappa Venkateshb, Ted M. Dawsonf, Kah Leong Limc,d,* a

Gene Regulation Laboratory, National Neuroscience Institute, 11 Jalan Tan Tock Seng, Singapore 308433, Singapore b Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673, Singapore c Neurodegeneration Research Laboratory, National Neuroscience Institute, 11 Jalan Tan Tock Seng, Singapore 308433, Singapore d Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117542, Singapore e Genome Institute of Singapore, 60 Biopolis Street, Singapore 138672, Singapore f Institute for Cell Engineering, Departments of Neurology and Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, United States Received 18 June 2004; received in revised form 9 September 2004; accepted 28 September 2004 Available online 15 December 2004 Received by A.J. van Wijnen

Abstract Mutations in the human parkin gene (huParkin) are the predominant genetic cause of familial parkinsonism. The huParkin locus, spanning about 1.4 Mb, is one of the largest in the human genome. Despite its huge size, huParkin codes for a rather short transcript of about 4.5 kb. To gain an insight into the structure, function and evolutionary history of huParkin, we have characterized the pufferfish [Fugu rubripes (Fugu)] ortholog of huParkin. A remarkable feature of the Fugu parkin gene (fuparkin) is its unusually compact size. It spans only about 4 kb and is thus 350-fold smaller than its human ortholog. The Fugu and human parkin genes are otherwise highly similar in their genomic organization and expression pattern. Furthermore, like human Parkin, Fugu parkin also functions as an ubiquitin ligase. These shared features between fuparkin and huParkin suggest that the physiological function and regulation of the parkin gene are conserved during the evolution of vertebrates. Conceivably, the compact locus of fuparkin could serve as a useful model to understand the transcriptional regulation of huParkin. D 2004 Elsevier B.V. All rights reserved. Keywords: Parkinson’s disease; Ubiquitin ligase; Transcriptional regulation; Dystrophin

1. Introduction Parkinson’s disease (PD) is a major neurodegenerative movement disorder affecting approximately 1% of the

Abbreviations: PD, Parkinson’s disease; RING, really interesting new gene; PACRG, parkin coregulated gene; ORF, open reading frame; PCR, polymerase chain reaction; RT, reverse transcription; RACE, rapid amplification of cDNA ends; TSS, transcriptional start site. * Corresponding author. Neurodegeneration Research Laboratory, National Neuroscience Institute, 11 Jalan Tan Tock Seng, Singapore 308433, Singapore. Tel.: +65 6357 7520; fax: +65 6256 9178. E-mail address: [email protected] (K.L. Lim). 0378-1119/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2004.09.022

elderly population worldwide (Siderowf and Stern, 2003). Although the etiology of PD remains poorly understood, the identity of several key genetic players whose mutations cause rare cases of familial parkinsonism has recently been elucidated (Lim and Lim, 2003). Of these, mutations in the parkin gene are currently recognized as the main contributor to familial parkinsonism (Kitada et al., 1998; Lucking et al., 2000). Emerging evidence also suggests a role for parkin in idiopathic PD and that parkin haploinsufficiency may be sufficient for the disease (West et al., 2002a; Foroud et al., 2003). To date, a wide range of parkin mutations, including several missense/nonsense/frameshift point mutations as well as exon deletion, duplication and triplication have

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been identified in parkin-related PD patients (Mata et al., 2004). It is currently unknown whether the heterogeneity in parkin mutations may in part be responsible for a wide age of onset (7–58 years) observed in patients with parkinassociated parkinsonism. The human parkin gene (huParkin), located on chromosome 6q26 (#6:161, 740, 081-163, 119, 211 May 04 Golden Path assembly), encodes a 465-amino-acid protein comprising a ubiquitin-like (UBL) domain at its Nterminus, a really interesting new gene (RING) box domain at its C-terminus and a unique middle segment that links the two domains (Kitada et al., 1998; Lim and Lim, 2003). The RING box domain of Parkin can be further subdivided into three consecutive cysteine-rich protein domains, namely, two C3HC4 RING fingers (RING 1 and 2) with a peculiar C6HC domain (IBR) separating them (Morett and Bork, 1999). At the extreme C-terminal end of the protein is a class II PDZ-binding motif bearing the amino acid sequence FDV that is important for the subcellular localization of Parkin within postsynaptic densities in the brain (Fallon et al., 2002). The consensus residues of the RING box domain and PDZ motif are identical in humans, rat and mouse (Gu et al., 2000; Fallon et al., 2002). Several groups have demonstrated that Parkin functions as an ubiquitin ligase associated with intracellular protein homeostasis and that the RING box domain is essential for its catalytic activity (Imai et al., 2000; Shimura et al., 2000; Zhang et al., 2000). Notably, many clinically relevant point mutations on Parkin occur on the RING box region. The huParkin locus, spanning about 1.4 Mb, is one of the largest in the human genome. It contains 12 exons and codes for a 4.5-kb transcript (Kitada et al., 1998; West et al., 2003). The largest known human gene to date is Dystrophin, which spans about 2 Mb. However, in contrast to huParkin, Dystrophin is composed of 79 exons and codes for a 13-kb transcript (Chelly et al., 1988; Ahn and Kunkel, 1993). Whereas the coding sequences of Dystrophin represents about 0.5% of the total length of the gene (Ahn and Kunkel, 1993; Pozzoli et al., 2003), the coding sequences of huParkin occupy only 0.1% of the gene length. It thus appears that huParkin has one of the highest ratio of noncoding to coding DNA lengths in the human genome. The role of the large introns which account for about 99.9% of huParkin is not known. Interestingly, the promoter region of huParkin is very short. Its 5V flanking gene, parkin coregulated gene (PACRG), resides just 204-bp upstream on the opposite strand (Asakawa et al., 2001; West et al., 2003). Given the close head-to-head linkages of the two genes, it is not surprising that their promoters overlap (West et al., 2003). It is possible that the two genes share some regulatory elements. However, despite its key role in familial parkinsonism, very little is known about the transcriptional regulation of huParkin. To gain an insight into the structure, function and regulation of huParkin, we have characterized the parkin

gene from the pufferfish, Fugu rubripes (Fugu). At 400 Mb, Fugu has the smallest vertebrate genome but codes for a similar gene repertoire to human (Venkatesh et al., 2000; Aparicio et al., 2002). The compact size of the Fugu genome is therefore useful in gene discovery as well as in understanding vertebrate gene structure and regulation. Typically, Fugu genes are about eightfold smaller compared to the human genes, in proportion to their genome sizes. However, we found that the size of the Fugu parkin gene (fuparkin) is dramatically compressed by about 350-fold compared to its human ortholog. Despite the huge reduction in the size, the structure, expression and biochemical function of fuparkin are similar to its human ortholog. The conserved features between the Fugu and human parkin genes suggest that the two genes may be similarly regulated.

2. Materials and methods 2.1. Database search and in silico analysis Sequences for the parkin gene from human, rat, mouse, Anopheles, Drosophila and C. elegans were retrieved from the UCSC Human Genome Browser at http://genome.ucsc. edu or the NCBI database at http://www.ncbi.nlm.nih.gov. As the parkin gene is only partially annotated in some of these species, we annotated the exons by iterative comparison of the genomic sequence with the cDNA sequence. To identify the genomic sequence of fuparkin, we searched the draft Fugu genome sequence at http://www.fugu-sg.org using the human Parkin protein sequence as the query. The genomic structure of fuparkin and its flanking genes were predicted using GENSCAN (http://genes.mit.edu/ GENSCAN.html). The exon–intron organization of fuparkin was confirmed by sequencing the cDNA amplified by reverse transcription (RT)–polymerase chain reaction (PCR) (see below). 2.2. 5V RACE, Cloning and RT–PCR analysis of Fugu parkin transcripts The cDNA templates used in these experiments were reverse-transcribed from the total RNAs of various Fugu tissues using the SMART rapid amplification of cDNA ends (RACE) cDNA Amplification kit (Clontech, USA). We used the 5V RACE approach to determine the transcription start site and the first exon of fuparkin. A nested PCR amplification of the cDNAs from Fugu brain was performed according to the manufacturer’s instruction. The two genespecific reverse primers used, 5V-GCAGAAGATCTGGCTGAACTCCCTGC-3V and 5V-CAGTGGCCTCTTCCTGCAGCTCTACG-3V, were derived from the putative second exon of fuparkin. The resulting PCR product was cloned into pGEM-T vector (Promega, USA) and sequenced completely. The full-length cDNA of Fugu parkin was amplified by PCR using the primers 5V-ACGAAGGTC-

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GA CG GG GC TGATGATCG-3V and 5V-TCC CAAAGATGCTGGGTCTGTCAACC-3V, and the resulting PCR product was cloned and sequenced. The restriction enzymes XhoI and ApaI were used to excise fuparkin cDNA from pGEM-T, and the cDNA was cloned into the mammalian expression vector, pCDNA3, in frame with a FLAG-epitope to generate pCDNA3-FLAG-fuparkin. The same pair of primers was used in RT–PCR to determine the expression pattern of Fugu parkin in different tissues. The PCR conditions comprised of an initial holding step at 95 8C for 2 min, followed by 35 amplification cycles of 95 8C for 30 s, 60 8C for 30 s and 72 8C for 2 min, and a final elongation step at 72 8C for 10 min. The quality of the Fugu cDNA was checked by PCR amplification of an actin fragment using the primers: ACTF: 5V-AACTGGGAYGACATGGAGAA-3V and ACTR: 5V-TTGAAGGTCTCAAACATGAT-3V. 2.3. Immunoprecipitation and Western blot HEK293 cells were transiently transfected with pCDNA3-FLAG-fuparkin in the absence or presence of pRK5-HA-Ubiquitin using the LipofectAMINE PLUS reagent (Invitrogen) according to the manufacturer’s instructions. Transfected HEK 293 cells were lysed in IP buffer containing 1% Triton-X 100, 10 Ag/ml aprotinin and 1 mM PMSF in PBS. Immunoprecipitations from the transfected cell lysates were performed with anti-FLAG antibody (Sigma) and protein G PLUS/Protein A-agarose (Oncogene Science) and then washed six times in lysis buffer.

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Immunoprecipitates or total cell lysates were analyzed by Western blot analysis with ECL detection reagents (Amersham-Pharmacia Biotech).

3. Results 3.1. Identification of Fugu parkin genomic sequence and transcription start site We used the human Parkin sequence to perform a TBLASTN search to identify the fuparkin sequence in the draft Fugu genome sequence. From the search, we were able to identify a single scaffold, Scaffold 289 (the third assembly), containing the fuparkin sequence. To determine the transcription start site (TSS) of fuparkin, we performed 5V RACE using primers specific to the putative second exon and obtained a single PCR product of 0.15 kb. We subsequently analyzed the sequence of the 5V RACE product from 23 independent clones and were able to identify consistently a TSS 41-bp upstream of the fuparkin start codon. Based on the sequence information, we mapped the boundaries of the first exon (Fig. 1A). We also isolated the full-length Fugu parkin cDNA by RT–PCR and used the derived sequence to determine the exon–intron structure of the entire fuparkin (Fig. 1B). The genomic organizations of fuparkin and huParkin are highly similar, except that exon 9 in fuparkin is split by an intron in humans (Figs. 1B and 3). However, the size of fuparkin is extremely compressed compared to its human ortholog. While huParkin spans 1.4-

Fig. 1. Genomic organization of the Fugu parkin gene. (A) Schematic diagram showing the sequence of fuparkin exon 1. The start codon is underlined. (B) Comparisons of the genomic structures of human and Fugu parkin genes. Exons are shown as rectangles in proportion to their sizes. Introns are represented by lines between exons, and their sizes (in kb) are indicated below. Dotted lines shows the splitting of fuparkin exon 9 into two exons in human.

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Mb genomic DNA, the entire fuparkin is only 4 kb, i.e., about 350-fold smaller than its human counterpart. This dramatic reduction in size of fuparkin relative to huParkin is largely due to its strikingly smaller introns (Fig. 1B). 3.2. Cross-species comparison of parkin gene size and genomic organization To gain an insight into the evolutionary history of the genomic organization and size of the parkin gene, we performed a comparative analysis of fuparkin with its orthologs from various vertebrate and invertebrate species. Examination of the parkin locus in these evolutionary distant organisms indicates that, while the length of the coding sequence is similar in all the organisms, there is an increase in the number of introns from invertebrates to vertebrates (Table 1). Whereas C. elegans, Anopheles and Drosophila parkin genes contain 4–6 introns, Fugu and mammalian parkin genes contain 10 and 11 introns, respectively. Furthermore, the introns of mammalian parkin genes are dramatically larger than those in invertebrates and Fugu, and consequently the mammalian parkin genes are much larger than others (Table 1). Interestingly, the parkin gene in chicken, which also contains 11 introns, appears to have an intermediate gene size between Fugu and mammals (Table 1). The smaller size of the parkin gene in invertebrates and Fugu compared to chicken and mammals suggests that the ancestral gene was small and that the gene enlarged considerably in the tetrapod lineage presumably due to the accumulation of repetitive sequences. 3.3. Promoter region of fuparkin To define the boundaries of the fuparkin promoter, we mapped its TSS by RACE and annotated the upstream gene based on homology to known genes. Inasmuch as PACRG is the immediate upstream gene to huParkin (West et al., 2003), we were curious to know if this arrangement is similar in the Fugu genome, relative to the human coordinates. We were able to identify through in silico Table 1 Comparison of parkin gene from various invertebrates and vertebrates Organisms

Size of parkin gene (kb)

Total size of exons (kb)a

Total size of introns (kb)

Number of introns

Human Mouse Rat Chicken Fugu Drosophila Anopheles C. elegans

1379 1190 1224 555b 3.50 1.85 1.75 1.72

1.395 1.392 1.395 1.389 1.446 1.446 1.467 1.158

1378 1189 1223 554b 2.058 0.407 0.282 0.559

11 11 11 11 10 5 4 6

a Total size of exons in each organism is calculated based on the respective length of their coding region. b Calculated size does not include the first exon and intron due to the lack of information.

analysis a single scaffold, Scaffold 395 (The third assembly), containing the Fugu ortholog of PACRG (fupacrg). Interestingly, fupacrg is not located upstream of fuparkin. Instead, a hypothetical gene is located about 800-bp upstream (Fig. 1A). The hypothetical gene, as predicted by GENSCAN, contains five exons which encodes a protein of 308 amino acids. The sequence of this putative protein is homologous to a rainbow trout EST (BX872772) and a zebrafish EST (BM036630). The hypothetical gene is therefore likely to be a true functional gene but appears to be fish-specific. Thus, in contrast to the highly compact size of fuparkin compared to huparkin, its intergenic region is much larger than that of the human gene. However, it is not clear if this expanded intergenic region arises as a result of a larger promoter region of the hypothetical gene located upstream of the fuparkin. Interestingly, a number of the Fugu genes on scaffold 289 appear to have orthologs on human chromosome 6. This conserved synteny provides additional evidence that the fuparkin identified by us is the exact ortholog of huparkin. Furthermore, unlike for many human–fugu gene pairs, there appears to be only one Fugu parkin gene, lending support to the notion that the genes are orthologs rather than paralogs. 3.4. Expression pattern of fuparkin Although mutations in huParkin causes the selective degeneration of nigral cells in the brain, its expression is not limited to brain tissues. Besides the brain, huParkin expression levels are also high in the heart and muscles but lower in tissues such as spleen, liver, thymus, lung and leukocytes (Kitada et al., 1998; West et al., 2003). We were interested to know if the differential expression pattern of parkin in various human tissues is conserved in Fugu. Examination of Fugu parkin mRNA distribution in a wide range of Fugu tissues by RT–PCR showed that, in addition to brain, fuparkin expresses in the heart, muscles, kidney, testis and ovary (Fig. 2). Lower levels of fuparkin expression are evident in the spleen and liver (Fig. 2). This pattern of fuparkin expression bears a striking resemblance to the expression pattern of its human counterpart and suggests that fuparkin and huParkin may have similar regulatory mechanisms. 3.5. Comparison of Fugu and human Parkin protein sequences Fugu and human Parkin protein sequences exhibit an overall identity of 53%. At their RING box region, the identity is much higher (61%) (Fig. 3). It is likely that the residues conserved between the Fugu and human Parkin proteins over the 450 million years of divergent evolution are functionally important. In support of this hypothesis, all the invariant residues in the RING box region known to be critical for parkin catalytic function are found to be conserved between Fugu and human. Furthermore, most

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Fig. 2. Expression pattern of Fugu parkin. RT–PCR results showing the expression pattern of Fugu parkin in various tissues relative to the expression pattern of h-actin.

of the amino acid residues in the human Parkin that are mutated in parkin-related PD patients are also conserved in Fugu parkin (Fig. 3). Interestingly, Fugu parkin lacks the PDZ motif (FDV) that is found at the carboxyl terminal of human, rat and mouse Parkin sequences. Presumably, Fugu parkin is incapable of mediating protein–protein interactions requiring the PDZ domain that otherwise take place in

mammalian cells, such as the binding of parkin with CASK/ Lin-2 (Fallon et al., 2002). 3.6. Functional analysis of Fugu parkin Inasmuch as a large number of the functionally important residues in the parkin protein are conserved between Fugu

Fig. 3. Protein sequence alignment of Fugu parkin and human Parkin. Parkin UBL and RING1–IBR–RING2 domains are underlined with dashed and solid lines, respectively. The PDZ motif bearing the sequence FDV is at the C-terminus of human Parkin. Conserved residues between the human and Fugu parkin protein are highlighted in black. Invariant residues typifying a RING or IBR domain are italicized and highlighted in bold. Human residues whose mutations are associated with PD are indicated with an asterisk. Arrowheads indicate the position of parkin exon–intron splice sites in Fugu (top) and human (bottom). The complete coding sequence of Fugu parkin has been deposited with GenBank (Accession no. AY507928).

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and human, we were interested to see if Fugu parkin also functions as an ubiquitin protein ligase like the human protein. To determine the biochemical function of Fugu parkin, we subcloned Fugu parkin cDNA into a mammalian expression vector in frame with a FLAG epitope and expressed the protein heterologously in HEK 293 cells. Anti-FLAG immunoblotting of HEK 293 lysate transfected with FLAG-tagged Fugu parkin reveals that the protein migrates as a 55-kD band, a size that is slightly higher than its 52-kD human counterpart, and correlates well with its predicted molecular weight (Fig. 4A). We investigated the potential ubiquitination property of Fugu parkin by transfecting FLAG-tagged Fugu parkin into HEK 293 cells in the presence or absence of HA-tagged ubiquitin to monitor its ability to perform self-ubiquitination. Parkin self-ubiquitination has been described in various reports and is a straightforward assay to analyze parkin catalytic property (Imai et al., 2000; Zhang et al., 2000). Anti-HA immunoblotting of parkin immunoprecipitated from lysates contain-

ing parkin coexpressed with ubiquitin reveals a ladder of anti-HA immunoreactivity that is consistent with parkin polyubiquitination (Fig. 4B). This staining pattern is not observed in parkin immunopreciptates prepared from lysates containing either parkin or ubiquitin alone (Fig. 4B). We obtained essentially similar results with the human Parkin control (Fig. 4B). Taken together, our results indicate that Fugu parkin, like its human ortholog, is a catalytically active ubiquitin ligase.

4. Discussion We have reported here the isolation and characterization of fuparkin, the Fugu ortholog of the familial parkinsonismassociated huParkin, and presented several features of fuparkin that supports its relevance and utility as a model to understand the structure and regulation of huParkin gene expression: (1) fuparkin is dramatically compressed, in

Fig. 4. Ubiquitin ligase activity of Fugu parkin (A) Anti-FLAG immunoblot of fractionated cell lysates prepared from untransfected HEK 293 cells or cells ectopically expressing either FLAG-tagged human parkin or Fugu parkin, as indicated. (B) Anti-FLAG or anti-HA immunoblots of parkin immunoprecipitates prepared from HEK293 cells expressing either FLAG-tagged parkin (human or Fugu) or HA-tagged ubiquitin alone or both. Lysates prepared from the transfected cells were also subjected to anti-FLAG or anti-HA immunoblotting to show their relative expression levels (INPUT).

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relation to its jumbo-size human counterpart; (2) huParkin and fuparkin exhibit highly similar genomic organization and tissue expression patterns, suggesting a shared regulatory mechanism; and (3) the human and Fugu parkin proteins are functionally analogous. Typically, Fugu genes are about 8- to 10-fold smaller than their human counterparts, in proportion to the ratio of their genome sizes (Venkatesh et al., 2000; Aparicio et al., 2002). The dramatic compression of fuparkin we found here, which is about 350-fold in relation to its human ortholog, is thus an unusual feature. The Fugu ortholog of the largest human gene, Dystrophin, is only 13-fold smaller (Pozzoli et al., 2003), while another large human gene, the 170-kb Huntingtin, is compressed by about sevenfold in the Fugu genome (Venkatesh et al., 2000). Although the Fugu ortholog of the 300-kb human h-amyloid precursor protein gene is also highly compacted (Venkatesh et al., 2000), its 30-fold reduction in size in the Fugu is less than a tenth we found here with fuparkin. Thus, fuparkin appears to be one of the most, if not the most, compressed Fugu gene relative to human orthologs. This raises the question whether the compactness of fuparkin is a result of intron compression in the Fugu lineage or intron expansion in the mammalian lineage. Our comparisons of the genomic structure of parkin from invertebrates, Fugu, chicken and mammals suggest that the small size of fuparkin is probably an ancestral state. Furthermore, the extra intron found in chicken and mammalian parkin genes with respect to fuparkin is the result of an intron bgainQ in the tetrapod lineage after it diverged from the fish lineage. The jumbo-size mammalian parkin genes are therefore likely to have arisen from a progressive expansion of intronic sequences in the tetrapod lineage. Lending support to this notion is the intermediate gene size of chicken parkin, which represents a lineage between fish and mammals. At present, it is not clear if the enlarged introns of mammalian parkin genes contribute to any unique function or regulation of the gene in mammals. However, it is generally believed that the large introns of mammalian genes act as a buffer zone to minimize the occurrence of lethal mutations in the coding sequences. In the case of huParkin, it is interesting that, despite the huge intron to coding sequence ratio, the coding sequence of the human gene is the target of numerous PD-causing mutations (Mata et al., 2004). A surprising feature of fuparkin is that, while conserved synteny is observed with huParkin, fupacrg is not located upstream of fuparkin. In mammals, both genes share a bidirectional promoter, and the genomic organization of huParkin/PACRG is reminiscent of a bacterial operon (West et al., 2003). Clearly, this is not the case in Fugu. Although very little is known about the transcriptional regulation of huParkin, it is apparent that many elements controlling mammalian parkin expression exist within the gene. Besides expressing in varying amounts in different tissues, parkin transcriptional level could also be modulated by several physiological cues and agents. For example,

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parkin mRNA and protein expression levels in cell culture models increase several folds during cellular unfolded protein stress (Imai et al., 2000). In rat striatum, the mRNA level of parkin also increases following acute and chronic administration of haloperidol, a dopamine-D2 receptor antagonist (Nakahara et al., 2001), but decreases following the administration of a neurotoxic dose of methamphetamine (Nakahara et al., 2003). The regulatory elements governing tissue-specific expression and responses to environmental or pharmacological modulators in these mammalian parkin genes, however, remain to be elucidated. The transcriptional regulation of huParkin is apparently important as emerging evidence indicates that variability in huParkin expression level is a risk factor in the development of PD (Hilker et al., 2001; West et al., 2002b). Although the core promoter region of huParkin has recently been identified, its structure did not reveal significant insights into the complexity of parkin transcriptional regulation (Asakawa et al., 2001; West et al., 2001), except that it contains a noncanonical myc binding site, which the transcription factor N-myc apparently binds to repress huParkin expression in the developing brain (West et al., 2004). Furthermore, inasmuch as PARCG resides just 204bp upstream from the start codon of huParkin (West et al., 2003), the resulting short intergenic interval between the two genes is likely to pose some structural constraints. In contrast, the situation is quite different with the very large size of the introns in the huParkin. As it is not uncommon for regulatory elements to be located in the introns, it is conceivable that additional parkin regulatory regions may potentially be located within its introns. However, to experimentally verify the existence of such elements in any of parkin gigantic introns would be a laborious and daunting task. In this light, the dramatically compact fuparkin, which is amenable to in vitro manipulation, offers an attractive alternative to uncover functionally relevant regulatory elements. We envisaged that the elucidation of these elements might facilitate the discovery of mutations of the noncoding regions of huParkin that give rise to parkin expression variability, an information that will be crucial for DNA diagnosis of PD patients for whom no mutations were found in the coding regions of parkin.

Acknowledgements This work was supported by grants from the National Medical Research Council (Y.W.P. and L.K.L.) and the National Healthcare/SingHealth Group (L.K.L.).

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