Understanding the role of ribosome-associated long non-coding RNA in cancers of the neural crest

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Whilst 85% of our genome is transcribed, only 4% is protein-coding. In fact, many non-coding RNAs have key roles in gene regulation and are termed long non-coding RNAs (lncRNAs). These exhibit far more tissue and developmental stage specific expression than mRNAs and are specifically enriched in the 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 but our recent Ribo-Ribo-Seq in human neuroblastoma cell line has revealed that many are cytoplasmic. We have discovered 278 cytoplasmic lncRNAs regulated during neuronal differentiation. 43% of these lncRNAs are associated with ribosomes but they themselves are not translated. We hypothesise that these ribosome-associated lncRNAs regulate mRNA translation in cancer. 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).Objectives1-Assess the importance of ribosome-associated lncRNAs to neuronal differentiation in neuroblastoma cell lines. 2-Determine the effect on mRNA translation by ribosome-associated lncRNAs. 3-Dissect molecular mechanisms of translational regulation by ribosome-associated lncRNAs. 4-Probe dysregulation of ribosome-associated lncRNAs in neuroblastoma. NoveltyThe project will take a range of cutting-edge and interdisciplinary approaches to understand the function of neuronal lncRNAs and their contribution to cancers of the nervous system 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.TimelinessOnly a small number of lncRNAs have been functionally characterised and the main focus has been on nuclear lncRNAs. Our recent work has identified this novel population of cytoplasmic lncRNAs which associate with ribosomes. The importance of these lncRNA-ribosome interactions has yet to be determined. RNA pull-down approaches have also recently been developed to isolate lncRNA-protein complexes from cells and tissues. Experimental Approaches-CRISPR/siRNA of lncRNA in neuroblastoma cell line 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 assays in neuroblastoma cell lines and primary cells-Analysis of publicly available cancer data on lncRNA expressed and dysregulation.