Background
Long non-coding RNAs (lncRNAs) form part of the human genome’s dark matter. The recent explosion in RNA-sequencing has revealed that lncRNAs exist in huge numbers. Compared to ~20,000 protein-coding genes, current estimates indicate humans possess ~15,000 lncRNA genes. Few of these lncRNAs have been characterised in detail, although many are associated with diseases, including cancer. The majority of lncRNAs are poorly conserved and expressed in a more cell specific manner than protein-coding mRNAs. For example, they are highly enriched in the human nervous system. Diseases of the nervous system are particularly associated with lncRNAs mis-regulation conditions e.g. Alzheimer’s disease.
The majority of characterised lncRNAs are nuclear and regulate ranscription. However, ~54% lncRNAs are localised in the cytoplasm, some of which function in development and differentiation. Recent evidence suggests that lncRNAs may interact with the translational machinery in the cytoplasm. We have discovered 278 cytoplasmic lncRNAs regulated during neuronal differentiation in a neuroblastoma cell line (Douka 2021). 43% of these lncRNAs are associated with ribosomes, although they are not translated. Such lncRNAs have been shown to regulate translation (Dimartino 2018). A subset of which regulate oncogenes, for example PHAROH lncRNA stimulates MYC translation in Hepatocellular carcinoma and represents a potential therapeutic target (Yu 2021).
In this project we will examine the hypothesis that these ribosome-associated lncRNAs affect mRNA translation in cancer. Unregulated mRNA translation is known to help drive the hallmarks of cancer (Song 2021). Therefore, understanding the contribution of lncRNAs to the control of mRNA translation in cancer such as neuroblastoma and other cancers arising in cells of neural crest origin is important for the development of novel therapeutic strategies. These studies could also identify prognostic biomarkers, which could be used as tools to select patients for lncRNA targeted treatment.
This project seeks to understand the molecular mechanisms of this translational regulation by lncRNAs during neuronal differentiation and the role these lncRNA play in cancers of the neural crest (e.g. neuroblastoma).
Objectives
- Assess the role of ribosome-associated lncRNAs during neuronal differentiation of neuroblastoma cell lines and patient-derived cultures.
- Determine the effect of ribosome-associated lncRNAs on mRNA translation.
- Dissect molecular mechanisms of translational regulation by ribosome-associated lncRNAs in normal cells.
- Probe dysregulation of ribosome-associated lncRNAs in neuroblastoma.
Novelty
The project will take a range of cutting-edge and interdisciplinary approaches to understand the function of neuronal lncRNAs and their contribution to cancers originating in the neural crest including neuroblastoma. By combining Next Generation Sequencing, structural biology, patient derived primary cells and cell biology, we will take a novel holistic view of neuronal lncRNA-protein biology.
Experimental Approaches
- CRISPR/siRNA of lncRNA in neuroblastoma cell lines and patient-derived neuroblastoma primary cultures
- Translation profiling by polysome gradients / Ribo-Seq
- Biochemical purification of lncRNA-ribosome complexes followed by cryo-EM
- Single molecule fluorescence microscopy of lncRNA in patient derived and culture neural crest cells
- Cell proliferation/ migration / single cell self-renewal assays in neuroblastoma cell lines and primary cells
- Analysis of publicly available cancer data on lncRNA expression and dysregulation.
https://aspdenlab.weebly.com/
https://biologicalsciences.leeds.ac.uk/heredity-development-disease/staff/19/dr-julie-aspden
https://astbury.leeds.ac.uk/people/dr-julie-aspden/
@RNA_julie
https://medicinehealth.leeds.ac.uk/medicine/staff/179/professor-sue-burchill
https://biologicalsciences.leeds.ac.uk/molecular-and-cellular-biology/staff/68/dr-juan-fontana
https://astbury.leeds.ac.uk/people/dr-juan-fontana-2/
@FontanaLab
Benefits of being in the DiMeN DTP:
This project is part of the Discovery Medicine North Doctoral Training Partnership (DiMeN DTP), a diverse community of PhD students across the North of England researching the major health problems facing the world today. Our partner institutions (Universities of Leeds, Liverpool, Newcastle, York and Sheffield) are internationally recognised as centres of research excellence and can offer you access to state-of the-art facilities to deliver high impact research.
We are very proud of our student-centred ethos and committed to supporting you throughout your PhD. As part of the DTP, we offer bespoke training in key skills sought after in early career researchers, as well as opportunities to broaden your career horizons in a range of non-academic sectors.
Being funded by the MRC means you can access additional funding for research placements, international training opportunities or internships in science policy, science communication and beyond. See how our current DiMeN students have benefited from this funding here: https://www.dimen.org.uk/blog
Further information on the programme and how to apply can be found on our website:
https://www.dimen.org.uk/how-to-apply
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