Journal of Molecular Biology
Identification of a Novel Gene Linked to Parkin via a Bi-directional Promoter
Introduction
Parkinson's disease (PD) is a common neurodegenerative disorder affecting greater than 2% of individuals over the age of 65.1 Recent studies have demonstrated the importance of genetic susceptibility in early-onset parkinsonism (onset<50 years)2 and late-onset sporadic PD.3., 4. The identification and isolation of genes that contribute to PD presents an opportunity to explore the molecular basis of the disease. Thus far, ten genetic loci have been implicated and five of the underlying genes have been identified.5 Four loci have been linked with autosomal recessive PD, for which two genes have been identified; parkin (PARK2) on chromosome 6q25-276 and DJ-1 (PARK7) on chromosome 1p36 (V. Bonifati, personal communication).
Mutations in the parkin gene (parkin, PARK2; OMIM 602544) are a common cause of autosomal-recessive juvenile (<20 years at onset) and early-onset parkinsonism.7., 8. Parkin is thought to function in the ubiquitin/proteasome system as a protein–ubiquitin E3 ligase, targeting substrate proteins for proteasomal degradation.9 Parkin is 465 amino acid residues long with an N-terminal ubiquitin-like domain and two RING domains. Parkin is unique, in that E3 ligases usually have a single RING domain for E2 cofactor binding. In addition, most RING-E3 ligases are multi-component protein complexes for which temporal and substrate specificity is conferred by F-box proteins.10 Parkin's structure and binding partner(s) in vivo are yet to be fully elucidated. Therefore, target substrate specificity may be more stringent than what is suggested by in vitro and co-transfection experiments. Currently, it is not known whether parkin associates with adapter proteins to mediate substrate recognition and/or downstream activity.
In addition to protein:protein interaction, knowledge of parkin gene expression and transcriptional regulation will help characterize parkin's cellular function. During our analysis of the parkin promoter, we identified a novel gene of unknown function, and we have named it parkin co-regulated gene (PACRG). Parkin and PACRG are linked in a head-to-head arrangement on opposite DNA strands and share a common 5′ flanking promoter region. Promoters possessing bi-directional functionality have been described11., 12. and these pairs of genes typically demonstrate overlapping, but not identical, expression patterns in different cell and tissue types.13 In many cases the gene products have similar functions or operate in a common pathway. For example, some enzyme complexes require tissue-specific, co-regulated expression of subunits. Here, we characterize the PACRG/parkin gene locus, and show that a common promoter regulates their expression. We postulate that PACRG may participate/interact with parkin in an, as yet, uncharacterized biological pathway.
Section snippets
Identification and characterization of PACRG
We compared human and mouse sequence of the parkin promoter derived from the Celera genome drafts† and observed a 52.8% homology of sequence extending 200 bp upstream of the human parkin transcription start site (TSS). Sequence further upstream showed an unexpected increase in conservation, with 76.8% homology observed between −200 bp and −400 bp of parkin exon 1. We hypothesized the possibility of a novel, overlapping gene. A BLAST analysis of the parkin promoter against the
Discussion
Mutations in the parkin gene are the most common cause of familial parkinsonism; understanding the cellular function and regulation of parkin will help elucidate the pathogenesis of idiopathic PD. Towards this end, we have previously identified the core promoter region of parkin within 250 bp upstream of parkin exon 1.14 Despite the fact that parkin is one of the largest genes in the human genome, we describe a novel gene with a TSS just 204 bp upstream of parkin, which has a similar
Cloning, 5′ RACE and Northern analysis of PACRG
PACRG exonic sequence was determined using human cDNA derived from heart, brain, testes and kidney, prepared from total RNA (Clontech, Palo Alto, CA) using the Superscript II RT-PCR kit (Invitrogen, Carlsbad, CA). Primers were designed (Invitrogen) to flank the predicted PACRG open reading frame (F 5′-TTCCAGACCTCCTGCTCACATCC-3′, R 5′-ACTCGGACTCCTGCTTCTGTCG-3′). PCR was carried out using a standard 60–50 °C touchdown program over 35 cycles. Products were analyzed on ethidium bromide-stained
Acknowledgements
We thank Dr John Hardy for useful discussions. We thank Dr Jamie Duckworth for help with in silico protein analysis and Minnie Schreiber for technical assistance. The Laboratories of Neurogenetics are supported in part by an NIH/NINDS Udall Center grant and the Mayo Foundation. P.J.L. is supported, in part, by a CJ Martin Fellowship from the Australian National Health and Medical Research Council. Substantia nigra used in this study was provided by the University of Miami Brain and Tissue Bank
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