Whilst 85% of our genome is transcribed, only 4% is protein-coding. In fact, many non-coding RNAs exist, some of which are similarly processed to mRNAs, termed long non-coding RNAs (lncRNAs). These exhibit far more tissue and developmental stage specific expression than mRNAs and are enriched in the nervous system and testes. Neurological disorders are particularly associated with lncRNAs mis-regulation conditions e.g. Alzheimer’s disease.
The majority of characterised lncRNAs are nuclear but our recent Ribo-Seq in human neuronal cells has revealed that many are cytoplasmic. We have discovered 278 cytoplasmic lncRNAs regulated during neuronal differentiation, ~40 of which show potential translation of small peptides. The line between coding and non-coding has become blurred and some lncRNAs are really mRNAs!
We have now shown that at least one lncRNA upregulated during neuronal differentiation is required for differentiation. This project seeks to understand the molecular mechanism of this lncRNA during neuronal differentiation and the role this lncRNA plays in neuronal cancers (e.g. neuroblastoma) and neurodegeneration.
1) Determine the molecular mechanism by which lncRNA regulates neuronal differentiation
2) Identify the interactors of the lncRNA (protein and RNA) and understand their structure
3) Assess the contribution of lncRNA to neuroblastoma and neuronal defects.
-Next Generation Sequencing to study lncRNA-ribosome interactions (Ribo-Seq)
-CRISPR/siRNA of lncRNA in neuronal cell lines
-Biochemical purification of lncRNA-protein complexes followed by mass spec and RNA-Seq
-Structural assessment of lncRNA-protein complex by cryo-EM
-lncRNA profiling of neuroblastoma patient derived cell lines
-single molecule fluorescence microscopy of lncRNA in neuroblastoma cell lines and patient derived cells
The project will take a range of cutting-edge and interdisciplinary approaches to understand the function of a neuronal lncRNA and its contribution to neuronal differentiation and disease. By combining Next Generation Sequencing, biochemistry, Cryo-EM, patient derived primary cells and cell biology, we are taking a novel holistic view of neuronal lncRNA-protein biology.
Only a small number of lncRNAs have been functionally characterized and the main focus has been on nuclear lncRNAs. In the last few years Ribo-Seq has revealed translation of a variety of non-canonical RNA regions, including lncRNAs, but translation events on lncRNAs remain controversial. The importance of these lncRNA-ribosome interactions has yet to be determined.
Our aim is to understand lncRNA function and deregulation in disease. This will improve our understanding of molecular pathways of neurodegeneration in which lncRNAs are key players. By using neural crest derived cells we will dissect the lncRNA role in self-renewing cells, differentiation and malignancy. By profiling our candidate lncRNAs in neuroblastoma patient cell lines this project has the potential to identify novel lncRNA biomarkers and molecular causes of both cancer and neurodegenerative diseases.
Aspden will provide lncRNA and genomics training, specifically Ribo-Seq. Aspden is academic co-lead for LeedsOmics initiative and contributes to a range of omics training opportunities e.g. bioinformatics for omics. Fontana and Astbury Centre will provide training on structural approaches, especially Cryo-EM. Hautbergue will provide training on neuronal systems, cell biology and patient-derived analysis.
Twitter @RNA_Julie http://aspdenlab.weebly.com/ https://biologicalsciences.leeds.ac.uk/molecular-and-cellular-biology/staff/19/dr-julie-aspden http://sitran.org/people/hautbergue/ https://biologicalsciences.leeds.ac.uk/molecular-and-cellular-biology/staff/68/dr-juan-fontana
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 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: http://www.dimen.org.uk/overview/student-profiles/flexible-supplement-awards
Further information on the programme can be found on our website: http://www.dimen.org.uk/
Aspden, J. L., Eyre-Walker, Y. C., Philips, R., Amin, U., Mumtaz, A. S., Brocard, M., Couso, J. P. Extensive translation of small ORFs revealed by Poly-Ribo-Seq. eLife 10.7554/eLife.03528 (2014).
Hautbergue, G. M., et al. SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits. Nature Communications 8:16063 (2017).
Cheen Euong Ang et al. The novel lncRNA lnc-NR2F1 is pro-neurogenic and mutated in human neurodevelopmental disorders eLife 2019;8:e41770 (2018)