The role of post-translational protein modifications (PTMs) in the regulation of RNP biomolecular condensates in health and motor neuron disease

Background. Eukaryotic cells contain a whole repertoire of microscopically visible, non-membrane-bound RNA-protein complexes - biomolecular condensates that are maintained through a combination of protein-protein, protein-RNA and RNA-RNA interactions. High concentration of molecules within these condensates makes these structures an ideal platform for the regulation of numerous processes related to mRNA localisation, translation, transport and turnover. 

Biomolecular condensates are abundant and prominent in neurons – large, long-lived cells. Therefore, it is not surprising that their dysfunction has been linked to the pathology of fatal neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Biogenesis, integrity, dynamics and ultimately, function of stress granule, paraspeckle, gem, P-body and speckle condensates are compromised in ALS making it a disease of disrupted cellular condensate network.

Phase separation properties of many proteins (primarily RNA-binding proteins, RBPs) and their ability to form/enter biomolecular condensates are regulated by post-translational modifications (PTMs), for example, arginine methylation. PTM dysregulation may therefore play a prominent role in diseases with a misbalance of the cellular condensate network, such as ALS.

Paraspeckles are prototypical biomolecular condensates formed by a long non-coding RNA NEAT1_2 that acts as a scaffold, and a set of proteins that stabilise this lncRNA and drive phase separation (paraspeckle proteins, PSPs), with the core ones being SFPQ, NONO and FET protein family members (FUS, TAF15 and NONO). These PSPs were found abnormally accumulated, mislocalised and/or aggregated across multiple ALS subtypes. Furthermore, mutations in PSPs can compromise paraspeckle integrity, and motor neurons, the affected neuronal population in ALS, are characterised by paraspeckle hyper-assembly. This suggests a reliance of this neuronal subtype on paraspeckles for survival and potentially depletion of this regulatory mechanism in PSP-linked ALS.

Project hypothesis. We hypothesise that PTMs of ALS-linked PSPs critically regulate and control their physiological and pathological phase separation.

Aim and objectives. The aim of this project is to comprehensively characterise the role of PTMs, in particular, arginine methylation and the antagonistic modification citrullination, of ALS-linked proteins – paraspeckle components – in physiological and pathological phase separation.

Objectives:

1. To identify PSPs whose regulation by PTMs is primarily responsible for paraspeckle clustering/separation, using mass-spectrometry coupled with super-resolution imaging.

2. To characterise the changes in the PSP and paraspeckle stability, dynamics, turnover and function upon manipulation of PSPs’ PTM profile, using pharmacological, molecular biology and super-resolution imaging approaches.

3. To characterise changes in the PTMs profiles in genetic models of ALS (including PSP-linked subtypes) and correlate these to paraspeckle structure and integrity.

Training and development opportunities for the student. This project will provide a unique opportunity to learn a well-rounded set of techniques for in-depth analysis of disease-linked proteins and their higher-order assemblies, including in vitro and in vivo analysis of biological phase separation, advanced proteomics and imaging, as well as use of cellular (genetic) disease models. The primary supervisor is a UKRI Future Leaders Fellow based at the Sheffield Institute for Translational Neuroscience (SITraN) – a centre with an outstanding reputation in training PhD students. The student will also spend a significant amount of time in the co-supervisors’ lab in the Department of Chemical and Biological Engineering (CBE) running an array of biochemistry/proteomics experiments and associated analyses, will attend lab meetings at both labs and present at internal seminars at both departments. Both labs are actively involved in curiosity-driven research of highest quality, and the multidisciplinary nature of the project will provide a space for research creativity thus offering an excellent training opportunity for an open-minded and flexible candidate.

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Please see this link for information on how to apply: https://www.sheffield.ac.uk/postgraduate/phd/apply. Please include the name of your proposed first supervisor and the title of the PhD project within your application.

Interviews will likely be held in April. Students must be able to start by October 2023. 

Applications are open to students from both the UK and overseas, though we note that due to funding constraints the availability of positions for students with overseas fee status will be more limited. We anticipate competition for these studentships to be very intense. We would expect applicants to have an excellent undergraduate degree in a relevant discipline. We would also expect applicants to have completed or be undertaking a relevant master’s degree to a similar very high standard (or have equivalent research experience).