Mutations in the DJ-1 gene are an extremely rare cause of a recessive form of Parkinson disease. Three homozygous point mutations and a deletion have been identified to segregate with disease in families while several other heterozygous mutations have been identified in studies of single early onset parkinsonism patients. The DJ-1 protein is part of a large family with homologues from humans to bacteria of uknown function. It is known, however, that DJ-1 can respond to oxidative stress in a very specific way, by the formation of a modified cysteine residue.? We have identified a novel potential function for DJ-1 of binding select groups of mRNA species. In collaboration with the Gorospe lab, NIA, we performed DJ-1 immunoprecipitation (IP) and microarray analysis of the associated RNA in three different systems. We started in M17 neuroblastoma cells then moved to mouse brains, using DJ-1 knockout mice as controls. We identified three consistent transcripts that we found in cells, namely mitochondrial genes, antioxidants and components of the PTEN (phosphatase and tensin homologue)/Akt1 pathway. ? We validated these interactions by RT-PCR and saw preferential amplification of a series of candidate transcripts, and we also used an antibody-independent technique, namely purification of 6xhistidine tagged proteins from transfected cells. In this last set of experiments, we were able to show that recessive mutant proteins are deficient in RNA binding activity.? We have also used cross-linking IP (CLIP) assays allowed us to isolate the bound RNA species and to then clone and sequence them. We recovered sequences that overlapped with those found in the array experiments. We also found that DJ-1 binds RNA directly at nanomolar concentrations. To understand what happens when DJ-1 binds RNA, we measured steady state protein levels for proposed RNA binding targets. We used paraquat exposure in cells and aging in mouse brain models of mild oxidative stress. The RNA levels for DJ-1 targets do not change but, under oxidative conditions, protein levels rise slightly in control cells or in wild type mouse brains. However, in the absence of DJ-1 (in knockout mice or knockdown lines), protein levels for the same targets tend to decrease. ? This leads to a hypothesis that DJ-1 allows translation under conditions of oxidative stress. We therefore used reporter assays for our candidate targets of DJ-1 RNA binding activity and showed that, for the specific target sequences, DJ-1 will repress translation.? Our current experiments are to try and replicate these results using human brain material. We are also examining whether the three specific pathways identified contribute to cellular dysfunction induced by lack of DJ-1.
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