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Precision Medicine DTP – Investigating disease causing mutations in PNPase- an approach to understand mitochondrial nucleic acid driven auto-inflammation


Project Description

Background

Mitochondrial diseases are a heterogeneous class of disorders often characterized by failure of mitochondrial function and loss of energy associated with respiratory chain complex deficiency. Mitochondrial functioning requires a complex interplay between nucleus and mitochondria as highlighted from the fact that 99% of the ~1200 mitochondrial proteome is nuclear encoded1. Nuclear gene products have diverse function in respiratory complex assembly, mtDNA maintenance and replication, gene expression, protein synthesis. Hence mitochondrial disorders can result from pathogenic variants in either the nuclear genome (nDNA) or mitochondrial genome (mtDNA). Due to this dual genetic control of mitochondrial function, these disorders can be inherited both in mendelian and maternal manner1.

It is reasonable to hypothesize that nuclear proteins implicated in mitochondrial biology may have diverse roles other than ascribed to mitochondrial function in energy generation alone.

In accordance, we have recently shown that key nuclear encoded mitochondrial RNA processing/maturation enzymes (SUV3, PNPase) have an unexpected novel role in preventing the formation of mitochondrial double-stranded RNA (mtdsRNA), a byproduct of mitochondrial bidirectional transcription2. mtdsRNA accumulation has deleterious pathological consequences as exemplified by inappropriate activation of innate immune activation and interferon response in patients carrying hypomorphic mutations in PNPase2. Underpinning this immune response is the unresolved issue of how mtdsRNA escapes the double-membrane compartments of mitochondria upon PNPase dysfunction thereby triggering such a potent innate immune response.

This PhD studentship will address the unresolved role of PNPase in mtdsRNA suppression and its escape crucial to innate immune response in PNPase patients using combination of genome engineering, super-resolution microscopy, dsRNA-seq and protein interactome approaches coupled with bioinformatics.

Aims

The first aim will be to engineer various disease-causing variants of PNPase using CRISPR knock-in that either reduce its protein levels or those that only abolish RNA degradation activity without affecting other functions so as to dissect out its role in mtdsRNA accumulation vs its escape. dsRNA-seq will be performed on these cells to define the effects of these mutations on mtdsRNA accumulation. These variants of PNPase will also be tagged endogenously at C-terminal with GFP using CRISPR approaches. This will enable to address PNPase localization, protein-protein interactome of various mutants compared to wildtype PNPase. Proteomic analysis of binding partners of the different mutant forms of the protein will be used to understand further the pathological consequences of individual mutations; this aspect of the project will involve training in the analysis of large datasets.

The second aim will be to develop various tools to monitor mtdsRNA by live cell imaging for a time lapse study using super-resolution microscopy on the above cell lines. This will include differential labelling of mitochondrial membranes to investigate its dynamics and ultrastructural features during mtdsRNA release. It will also emphasize on identifying involvement of various mitochondrial channels or pores in such a process. This is a powerful approach that we have previously applied to understand mitochondrial DNA release in apoptotic stimuli3.

This will provide deeper understanding of PNPase associated disease mechanisms as a way to uncover its role in mitochondrial dysfunction and innate immune activation.

Training Outcomes

We have techniques in place so the student will benefit from being able to hit the ground running, but also from training in, for example CRISPR/Cas9, super-resolution microscopy, high-throughput approaches like dsRNA-seq and proteomic data analysis (including the use of R). In the later stages of the PhD the student will be encouraged to seek out collaborations within UoE groupings, allowing them to take ownership of the project and to develop their independence within a secure background of newly developed expertise.

This MRC programme is joint between the Universities of Edinburgh and Glasgow. You will be registered at the host institution of the primary supervisor detailed in your project selection.

All applications should be made via the University of Edinburgh, irrespective of project location. For those applying to a University of Glasgow project, your application along with any supporting documents will be shared with University of Glasgow.

http://www.ed.ac.uk/studying/postgraduate/degrees/index.php?r=site/view&id=919

Please note, you must apply to one of the projects and you must contact the primary supervisor prior to making your application. Additional information on the application process is available from the link above.

For more information about Precision Medicine visit:
http://www.ed.ac.uk/usher/precision-medicine

Funding Notes

Start: September 2020

Qualifications criteria: Applicants applying for a MRC DTP in Precision Medicine studentship must have obtained, or will soon obtain, a first or upper-second class UK honours degree or equivalent non-UK qualification, in an appropriate science/technology area.
Residence criteria: The MRC DTP in Precision Medicine grant provides tuition fees and stipend of at least £15,009 (RCUK rate 2019/20) for UK and EU nationals that meet all required eligibility criteria.

Full eligibility details are available: View Website

Enquiries regarding programme:

References

1. Lightowlers RN, Taylor RW, Turnbull DM: Mutations causing mitochondrial disease: What is new and what challenges remain? Science. 2015 Sep 25;349(6255):1494-9.

2. Dhir A, Dhir S, Borowski L, Jimenez L, Teitell M, Rötig A, Crow YJ, Rice GI, Duffy D, Tamby C et. al.: Mitochondrial double stranded RNA triggers antiviral signalling in humans. Nature. 2018 Aug;560 (7717): 238-242.

3. Riley JS, Quarato G, Cloix C, Lopez J, O'Prey J, Pearson M, Chapman J, Sesaki H, Carlin LM, Passos JF, Wheeler AP, Oberst A, Ryan KM, Tait SW: Mitochondrial inner membrane permeabilisation enables mtDNA release during apoptosis. EMBO J. 2018 Sep 3;37(17).

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