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

了解核糖体相关长非编码 RNA 在神经嵴癌中的作用

• 批准号: 2879751
• 金额: --
• 依托单位: University of Leeds
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2023
• 资助国家: 英国
• 起止时间: 2023 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2879751
• 关键词: Understanding; role; ribosome; associated; long

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.

虽然我们的基因组 85% 是转录的，但只有 4% 是蛋白质编码的。事实上，许多非编码 RNA 在基因调控中发挥着关键作用，被称为长非编码 RNA (lncRNA)。它们比 mRNA 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富集。神经系统疾病尤其与 lncRNA 失调相关。阿尔茨海默病。大多数特征化的 lncRNA 是细胞核的，但我们最近在人神经母细胞瘤细胞系中进行的 Ribo-Ribo-Seq 表明，许多 lncRNA 是细胞质的。我们发现了 278 个细胞质 lncRNA 在神经元分化过程中受到调节。这些 lncRNA 中有 43% 与核糖体相关，但它们本身不被翻译。我们假设这些核糖体相关的 lncRNA 调节癌症中的 mRNA 翻译。该项目旨在了解神经元分化过程中lncRNA翻译调节的分子机制，以及这些lncRNA在神经嵴癌（例如神经母细胞瘤）中发挥的作用。 目标1-评估核糖体相关lncRNA对神经母细胞瘤细胞神经元分化的重要性线。 2-确定核糖体相关 lncRNA 对 mRNA 翻译的影响。 3-剖析核糖体相关 lncRNA 翻译调控的分子机制。 4-探针神经母细胞瘤中核糖体相关 lncRNA 的失调。新颖性该项目将采用一系列前沿和跨学科的方法来了解神经元lncRNA的功能及其对神经系统癌症（包括神经母细胞瘤）的贡献。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-蛋白质生物学采取新颖的整体观点。及时性仅对少数 lncRNA 进行了功能表征，主要关注点是核 lncRNA 。我们最近的工作发现了这种与核糖体相关的新型细胞质 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 Pull-down 方法，用于从细胞和组织中分离 lncRNA-蛋白质复合物。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物中lncRNA的CRISPR/siRNA-通过多核糖体梯度/Ribo-Seq进行翻译分析-lncRNA-核糖体复合物的生化纯化，然后进行冷冻-EM-lncRNA的单分子荧光显微镜检查患者来源和培养的神经嵴细胞 - 神经母细胞瘤细胞系和原代细胞中的细胞增殖/迁移测定 - 分析公开的有关 lncRNA 表达和表达的癌症数据失调。