ESR 11 - Vanessa Xavier
Mitochondrial RNA granule associated proteins: investigating their roles in mtDNA gene regulation and expression
Principal Investigator and Sending Institution:
Prof. Jean Claude Martinou, University of Geneva, Switzerland
Principal Investigator and Host Institution:
Dr. Ashish Dhir, University of Edinburgh, Scotland
14 January - 20 March 2020
Report - In my home lab, I had observed that throughout tumorigenesis, after the induction of oncogenes, dsRNA foci could be...
I decided to carry out my secondment at the laboratory of Dr Ashish Dhir at the Insitute of Genetics and Molecular Medicine (IGMM) in Edinburgh, Scotland. The IGMM is a part of the University of Edinburgh and works closely with several research institutes. Dr Dhir recently was awarded the Chancellor’s Fellowship to set up his new group. He recently discovered a novel link between the release of mitochondrially derived dsRNA and the initiation of an inappropriate immune response, following a defect in the exoribonuclease PNPase. (Nature, 2018) His work has laid the foundation for further investigation into the mitochondrion’s role in innate immunity and more fundamentally, the existence of the dsRNA species which derives from the bi-directional transcription of the mitogenome.
As a REMIX fellow in Prof. Jean-Claude Martinou’s group at the University of Geneva, my main research topic involves the study of the Mitochondrial RNA granules (MRGs). Named as such for the membraneless complex of mitochondrial RNA and proteins within the mitochondrial matrix. The MRGs are visualised by immunofluorescence as punctate structures within the mitochondrial network, resembling beads on a string. After the publication of Dr.Dhir’s work, the presence of dsRNA foci, visualised by an antibody specific for dsRNA, opened up a new avenue of investigation into RNA granules in the mitochondria. We have collaborated closely with him since then, establishing preliminary data on MRGs and dsRNA foci. Therefore, although Dr.Dhir isn’t part of the consortium, fortunately, I was able to fulfil my secondment in his laboratory in Edinburgh.
We decided to study dsRNA from 2 different angles. Firstly, we wanted to establish the localisation of dsRNA foci within the scheme of mitochondrial gene expression. As we had already established them to be different from MRGs but always closely associated, and that an accumulation of dsRNA caused a mitochondrial translation defect downstream, we concluded that it was not merely degraded away. With Dr.Dhir, we developed an IP-qPCR experiment to pulldown newly synthesized mt-RNA and target processed and unprocessed genetic regions of the RNA. I was also able to perform super-resolution Structured Illumination Microscopy (SIM), with 4 different fluorophores to visualise the different RNA and protein components of the MRGs and dsRNA that I could not achieve with STED microscopy, due to its inherent technical limitations. These data have lent more evidence to our hypothesis on the place of dsRNA within gene expression.
Secondly, we also investigated the correlation of dsRNA accumulation in cancer cells and the innate immune response. In my home lab, I had observed that throughout tumorigenesis, after the induction of oncogenes, dsRNA foci could be visualised in cells, where there were none seen in primary cells. Although I had initially chalked this up to the increase in transcription that accompanied the tumourigenic process, I wanted to study if the changing immunological state of the cell had a role to play. Dr.Dhir is affiliated with Prof. Yannick Crow’s group, whose studies disorders associated with an enhanced type I interferon signalling. Therefore, I was able to carry out experiments looking in the innate immunity pathways that are triggered by nucleic acid-sensing within the cells. The results of these experiments have given us a few hypotheses to further work on.
Apart from the bench, my secondment was an opportunity to experience Edinburgh through its history and culture. However, the Scottish stereotype of constant bad weather exists for a reason! Although I was not brave enough to fend off the strong wind, rain and snow, I was constantly mesmerised by the dramatic backdrop that it served to highlight the beautiful architecture and impressive mountains that surround Edinburgh. I also knew about the morbid history of the city, especially that of grave robbers who would dig up fresh corpses to sell to the Faculty of Medicine at the University of Edinburgh for their anatomy lessons. This led to people building literal cages around their graves to prevent their loved ones from falling victim to this crime, which can still be seen in older graveyards. On a lighter note, I also visited a Gin distillery and a Whisky tasting experience where I won a contest for identifying the highest number of whiskies correctly and snagged a bottled of a personalised blended Scotch!
Altogether, the IGMM is a highly collaborative environment which fortunately has complementary facilities that allowed me to carry out experiments that I could not have done as easily in Geneva. I was able to present my work in front of a new audience and acquaint myself with the scientific community that exists in Scotland and is producing impressive work. Most of all, I am grateful to Dr.Dhir for firstly agreeing to house me in his lab. He was and still is, extremely generous with ideas, experimental materials, advice and mentorship that was extremely encouraging and has left me with renewed vigour to continue my work in Geneva.