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EASTBIO Revealing nuclear architecture – Multiplexed labelling of RNA, DNA and proteins using super-resolution microscopy


Project Description

A PhD studentship is available in the group of Dr Annamaria Lilienkampf (School of Chemistry, University of Edinburgh). The project will be co-supervised by Prof Nick Gilbert (MRC Institute of Genetics & Molecular Medicine, University of Edinburgh)

Project Details:
The aim of this project is to develop and use bioorthogonal tools to simultaneously label DNA and RNA, and to visualise them together with proteins, namely SAF-A, using super-resolution microscopy. These new tools will help to answer fundamental biological questions of how DNA, RNA and SAF-A interact in the cell nucleus and how SAF-A levels influence nuclear architecture in neurons. This truly multidisciplinary PhD project will provide skills in organic chemistry (e.g. synthesis of fluorophores, modified nucleosides, and tetrazines), cell culture and molecular biology techniques, as well as super-resolution imaging.
SAF-A (Scaffold Attachment Factor A, also known as HNRNPU) is a key nuclear protein with gene mutations and aberrant expression linked to neurological disorders such as epilepsy and autism.1 We have recently shown that SAF-A oligomerises in the presence of ATP and chromatin-associated RNA form a dynamic nuclear mesh, which maintains euchromatin in a decompacted configuration, with low SAF-A protein levels causing genomic instability.2 To fundamentally understand how SAF-A interacts with DNA and RNA3 to form a nuclear mesh and how aberrant protein scaffold levels affect nuclear architecture, it is necessary to simultaneously label and visualise SAF-A, RNA and DNA at high resolution.

The Lilienkampf and Gilbert Labs have shown that RNA and DNA can be independently labelled using bioorthogonal chemistry tools. Metabolic labelling of RNA and DNA with chemically modified Uridine and Deoxyuridine (EdU), respectively, enabled subsequent visualisation by wide-field light microscopy using bioorthogonal tetrazine/alkene and azide/alkyne “click-chemistries” with fluorescently tagged probes whereas SAF-A can be labelled using a SNAP-tag or with fluorescent antibodies (methods available in the Gilbert lab). In this PhD project, we will now develop an imaging platform to enable simultaneous detection of RNA, DNA and proteins by using super-resolution microscopy.

To investigate nuclear organisation and kinetics, metabolically tagged DNA and RNA will be fluorescently labelled using azide and tetrazine-based fluorescent probes alongside a fluorescent antibody for SAF-A. We will investigate a combination of different fluorophores.

Once successful multiplexed labelling is achieved in normal cells using wide field or confocal microscopy, the imaging platform will be transferred for super-resolution imaging, using direct stochastic optical reconstruction microscopy (D-STORM) taking advantage of methods we have recently developed. Finally, these novel imaging tools will be used to investigate the localisation and interaction of SAF-A, RNA and DNA in neuronal cells (such as LUHMES) and ultimately used to investigate how SAF-A depletion (by means of auxin-inducible degron system or by RNA interference) affects nuclear architecture (and DNA damage response) in neurons.

Application Process:
To apply for an EASTBIO PhD studentship, follow the instructions below:

1) Check FindaPhD for our available projects and contact Dr Annamaria Lilienkampf before you apply.

2) After you have discussed the projects of interest to you with the project supervisors, download and complete our Equality, Diversity and Inclusion survey and then fill in the EASTBIO Application Form and submit to each of your proposed projects as per the instructions in the project adverts.

3) Send the EASTBIO Reference Form to your two academic/professional referees, and ask them to submit as specified on the project adverts.

4) If you are nominated by the supervisor(s) of the EASTBIO PhD project you wish to apply for, they will provide a Supervisor Support Statement.

5) We anticipate that our first set of interviews will be in the week commencing 10th February 2020 with awards made the following week.

If you have further queries about the application/recruitment process please email EastBio.

Please ask your referees to submit your references directly to Dr Annamaria Lilienkampf

The School of Chemistry holds a Silver Athena SWAN award in recognition of our commitment to advance gender equality in higher education. The University is a member of the Race Equality Charter and is a Stonewall Scotland Diversity Champion, actively promoting LGBT equality. The University has a range of initiatives to support a family friendly working environment. See our University Initiatives website for further information. University Initiatives website: https://www.ed.ac.uk/equality-diversity/help-advice/family-friendly

Funding Notes

This 48 month fully-funded PhD position is supported through the BBSRC EastBio Doctoral Training Partnership. Applicants must hold a first or upper second-class UK honours degree or equivalent and must meet RCUK residency requirements. More information can be found here: View Website

References

1. Bramswig, N.C., Lüdecke, HJ., Hamdan, F.F. et al. Human Genetics, 2017, 136: 821
2. Nozawa, R-S., Boteva, L., Soares, D. C., Naughton, C., Dun, A. R., Buckle, A., Ramsahoye, B., Bruton, P. C., Saleeb, R. S., Arnedo, M., Hill, B., Duncan, R. R., Maciver, S. K., Gilbert, N., Cell, 2017, 169 (7), 1214–1227.e18
3. Nozawa, R-S., Gilbert, N. Trends in Cell Biology, 2019, 29 (3), 201¬–211

How good is research at University of Edinburgh in Chemistry?
(joint submission with University of St Andrews)

FTE Category A staff submitted: 43.30

Research output data provided by the Research Excellence Framework (REF)

Click here to see the results for all UK universities

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