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

虽然我们85%的基因组是转录的，但只有4%是蛋白质编码的。事实上，许多非编码RNA在基因调控中起着关键作用，被称为长非编码RNA(Long Non-Coding RNAs，LncRNAs)。它们比mRNAs表现出更多的组织和发育阶段特异性表达，并且在神经系统中特异性地丰富。神经系统疾病尤其与lncRNAs调节不当有关，例如阿尔茨海默病。大多数特征性的lncRNA是核的，但我们最近在人类神经母细胞瘤细胞系中的Ribo-Ribo-Seq揭示了许多是细胞质的。我们已经发现了278个在神经元分化过程中受到调控的细胞质lncRNAs。其中43%的lncRNA与核糖体相关，但它们本身并不被翻译。我们假设这些核糖体相关的lncRNAs调节癌症中的mRNA翻译。本项目旨在了解在神经元分化过程中lncRNAs翻译调控的分子机制，以及这些lncRNA在神经脊癌(如神经母细胞瘤)中所起的作用。目的1-评估核糖体相关的lncRNAs在神经母细胞瘤细胞系中对神经元分化的重要性。2-确定核糖体相关的lncRNAs对mRNA翻译的影响。3-剖析核糖体相关lncRNAs翻译调控的分子机制。神经母细胞瘤中核糖体相关lncRNAs的4探针调控异常。该项目将采用一系列尖端和跨学科的方法来了解神经元lncRNAs的功能及其在包括神经母细胞瘤在内的神经系统癌症中的作用。通过结合下一代测序、结构生物学、患者来源的原代细胞和细胞生物学，我们将对神经元lncRNA-蛋白质生物学有一个新的整体观点。时间上，只有少量的lncRNA被确定了功能特征，主要集中在核lncRNA上。我们最近的工作已经确定了与核糖体相关的这一新的细胞质lncRNA群体。这些lncRNA-核糖体相互作用的重要性尚未确定。最近还开发了RNA下拉方法来从细胞和组织中分离出lncRNA-蛋白质复合体。实验方法-神经母细胞瘤细胞系和患者来源的神经母细胞瘤原代培养细胞中lncRNA的CRISPR/siRNA-多聚体梯度翻译图谱/Ribo-Seq-lncRNA-核糖体复合体的生化纯化，随后冷冻-EM-单分子荧光显微镜对患者来源和培养的神经脊细胞中的lncRNA-cRNA的研究-神经母细胞瘤细胞系和原代细胞中的细胞增殖/迁移分析-关于lncRNA表达和调控失调的公开癌症数据的分析。

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.