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Precision Medicine DTP - Mitochondrial double-stranded RNA driven innate immune activation in mitochondrial cytopathies


College of Medicine and Veterinary Medicine

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Prof Y Crow , Dr A Dhir , Dr S Tait , Dr Tamir Chandra No more applications being accepted Competition Funded PhD Project (Students Worldwide)

About the Project

Background

Recognition of Pathogen-associated nucleic acid (NA) species as ‘foreign’ represents the primary mechanism by which mammalian cells launch a type I interferon (IFN)-mediated innate immune response against viral and bacterial infections. Given the biochemical universality of nucleic acids and their involvement in driving the host immune response, a challenge exists to ensure that self-derived endogenous NA is not misrepresented as foreign. Regulatory mechanisms must exist to maintain cellular and organismal homeostasis, while genetic and environmental alteration of these regulatory processes can lead to self-derived sterile inflammation and inflammatory disorders - including the monogenic type I interferonopathies, and more common autoimmune phenotypes such as systemic lupus erythematosus (SLE)1. Mitochondria, as evolutionary relics of bacteria, are a potential source of immunogenic, self-derived, nucleic acids capable of triggering an antiviral cytokine response in humans. We are interested in the axis of NA metabolism and innnate immunity in monogenic mitochondrial cytopathies, wherein alteration of NA metabolism may be an underlying cause of inappropriate innate immune stimulation.

In accordance with this possibility, we have recently shown that two key nuclear encoded mitochondrial RNA processing/maturation enzymes (SUV3, PNPase) have an unexpected, and 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 innate immune activation and the induction of an 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.

This PhD studentship will elucidate disease mechanism(s), consequent upon mtdsRNA accumulation and its cytosolic escape, that underlies the innate immune activation observed in patients with PNPase deficiency and other related mitochondrial cytopathies. This will involve genome engineering, super-resolution microscopy, RNA-seq and protein interactome approaches coupled with bioinformatic analysis.

Aims

The first aim will be to engineer various disease-causing variants of PNPase, using CRISPR knock-in that either reduce protein levels or selectively abolish RNA degradation activity, as a way to dissect out the phenomena of mtdsRNA accumulation and mitochondrial escape. dsRNA-seq and immunofluorescence, coupled with innate immune activation assays, will be performed to define the effect of mutations on mtdsRNA accumulation. Variants of PNPase will also be tagged endogenously at C-terminal with GFP using CRISPR-Cas9 approaches, enabling us to understand the submitochondrial localization of PNPase and perform protein interactome studies of mutants compared to wildtype. Proteomic analysis of binding partners of various mutants of PNPase will further help us determine the pathological consequences of individual mutations; this aspect of the project will involve training in the analysis of large datasets. These results will be validated in patient cell lines.

The second aim will be to develop tools to understand the mechanism of mtdsRNA release upon PNPase dysfunction. This will include the differential labelling of mitochondrial membranes to investigate its dynamics and ultrastructural features using super-resolution microscopy and live cell imaging. There will be an emphasis here on identifying the mitochondrial channels or pores involved in mtdsRNA release. This is a powerful approach that we have previously applied to understand mitochondrial DNA release to apoptotic stimuli3.

Overall, the project will provide a deeper understanding of PNPase associated disease mechanisms as a way to defining the link between 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. Training will be provided in 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 2021

Qualifications criteria: Applicants applying for an 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. The MRC DTP in Precision Medicine grant provides tuition fees and stipend of at least £15,285 (UKRI rate 2020/21).

Full eligibility details are available: http://www.mrc.ac.uk/skills-careers/studentships/studentship-guidance/student-eligibility-requirements/

Enquiries regarding programme: [Email Address Removed]

References

1. Uggenti C, Lepelley A, Crow YJ. Self-Awareness: Nucleic Acid-Driven Inflammation and the Type I Interferonopathies. Annu Rev Immunol. 2019 Apr 26;37:247-267. doi: 10.1146/annurev-immunol-042718-041257. Epub 2019 Jan 11. PMID: 30633609

2. Dhir A, Dhir S, Borowski L, Jimenez L, Teitell M, Rötig A, Crow YJ, 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, 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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