Primary Supervisor: Dr Mariaelena Repici, Life & Health Sciences
Secondary supervisor: Professor Andrew Devitt, Dr. Ivana Milic
PhD project title: Unravelling the role of DJ-1 in cellular RNA dynamics: relevance for neurodegeneration
University of Registration: Aston University
Mutations in the gene encoding DJ-1 are associated with autosomal recessive forms of Parkinson’s disease (PD). DJ-1 plays a role in protection from oxidative stress, but how it functions as an “upstream” oxidative stress sensor and whether this relates to PD is still unclear. Intriguingly, DJ-1 may act as an RNA binding protein associating with specific mRNA transcripts in the human brain. We previously reported that the yeast DJ-1 homolog Hsp31 localizes to mRNA granules known as stress granules (SGs) and P-bodies after glucose starvation, suggesting a role for DJ-1 in RNA dynamics. SGs are cytoplasmic aggregates that represent the morphological consequence of an mRNA triage process triggered by environmental stresses (Anderson and Kedersha, 2008). These structures are characterized by the presence of the translationally silent 48S preinitiation complex (mRNA transcripts, 40S ribosomal proteins, eIF3, eIF4A, eIF4B, eIF4G and eIF4E and PABP-1) and represent the physical place within the cytoplasm of stressed cells where the fate of mRNA transcripts is decided. In our recently published article in Molecular Neurobiology (Repici et al., 2019) we show that DJ-1 interacts with several SG components in mammalian cells and localizes to SGs and P-bodies upon induction of stress. Moreover, we find that subpopulations of mRNAs which interact with DJ-1 localize to SGs after stress, suggesting that DJ-1 may target specific mRNAs to mRNP granules. Notably, we demonstrate that DJ-1 associates with SGs arising from N-methyl-D-aspartate (NMDA) excitotoxicity in primary neurons and parkinsonism-related toxins in dopaminergic cell cultures. Our results indicate that DJ-1 is associated with cytoplasmic RNA granules arising during stress and neurodegeneration, providing a possible link between DJ-1 and RNA dynamics which may be relevant for PD pathogenesis and fits within the emerging area of RNA metabolism and neurodegenerative diseases.
This project aims at further investigating the role of DJ-1 in RNA dynamics, to elucidate how DJ-1 interactions with mRNAs changes in pathological versus physiological conditions. This will be achieved by a) exploring the DJ-1/RNA interactome and its role in translation, b) studying the compartmentalization of DJ-1 within stress granules and c) determining the mRNA populations targeted by DJ-1 to SGs. Ultimately, such analyses will further elucidate how DJ-1 loss of function leads to PD, providing important insight into the molecular pathogenesis of this disorder and potentially informing novel therapeutic strategies.
The project will involve:
- Genomic approach to study the RNA populations in neuronal cells that interact with DJ-1 in different stress conditions. The mouse dopaminergic neuronal SN4741 cell line will be exposed to hyperosmotic shock, oxidative stress or MPP+, and will be lysed in polysome lysis buffer. RNAs interacting with DJ-1 will be purified by co-immunoprecipitation as in Repici et al. 2109, and RNA sequencing will be carried out. Gene ontology and related bioinformatics approaches will be employed to identify functional groups arising from the various stimuli, which will inform the signalling pathways regulated by DJ-1.
- Use of U-2 OS cells expressing a GFP-G3BP1 fusion protein to isolate SG cores after different type of stress (hyperosmotic shock, oxidative stress, MPP+). Immunoblotting will be performed on the SG cores to look for total DJ-1, as well as oxidized forms of DJ-1. This will clarify whether DJ-1 acts as an RNA shuttle protein in the outer shell of SGs or as a core SG component. Mass spectrometry will be used for the analysis of SG components to identify DJ-1 oxidation state and post translational modifications.
- RNA in situ hybridization of the DJ-1 interacting mRNAs in stress conditions will be performed using using the RNAScope Multiplex Assay and combined with immunofluorescence for SG markers in relevant cells. This work will clarify whether candidate mRNAs being regulated by DJ-1 are being sequestered in SGs during stress conditions.
- Anderson and N. Kedersha. Trends Biochem Sci 33 (3), 141 (Mar 2008).
- Repici M, Hassanjani M, Maddison DC, Garção P, Cimini S, Patel B, Szegö ÉM, Straatman KR, Lilley KS, Borsello T, Outeiro TF, Panman L, Giorgini F. The Parkinson's Disease-Linked Protein DJ-1 Associates with Cytoplasmic mRNP Granules During Stress and Neurodegeneration. Mol Neurobiol. 2019 Jan;56(1):61-77.
BBSRC Strategic Research Priority: Understanding the Rules of Life: Neuroscience and behaviour
Techniques that will be undertaken during the project:
- Cell culture
- RNA purification and RNA sequencing
- Protein co- immunoprecipitation and western blot
- RNA in situ hybridization
- Immunofluorescence and confocal microscopy
- Image analysis
- Mass spectrometry
Contact: Dr Mariaelena Repici, Aston University