Understanding the regulatory language of RNA localization

了解 RNA 定位的调控语言

• 批准号: 9796036
• 负责人: Jefferson Matthew Taliaferro
• 金额: $ 38.21万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-15 至 2024-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9796036
• 关键词: Apical; Biochemical; Biological Assay; Cell Fractionation; Cell physiology; Cellular Morphology; Defect; Development; Drosophila genus; Elements; Environment; Epithelial Cells; Eukaryotic Cell; Exons; Gene Expression; Genetic Transcription; Genome engineering; Human; Intestines; Knowledge; Language; Language Tests; Lead; Light; Location; Mammalian Cell; Mediating; Methods; Modeling; Molecular; Neuroglia; Neurons; Null Lymphocytes; Organism; Outcome; Phenotype; Post-Transcriptional Regulation; Process; RNA; RNA Binding; RNA Decay; RNA Sequences; RNA Splicing; RNA-Binding Proteins; Regulation; Reporter; Site; System; Techniques; Time; Transcript; Untranslated RNA; Work; cell type; combinatorial; experimental study; high throughput screening; innovation; nervous system disorder; next generation sequencing; trafficking; transcriptome

SUMMARY Eukaryotic cells contain within them a myriad of spatially distinct sites that serve a variety of functions. To facilitate this organization, eukaryotic gene expression is routinely spatially regulated through the trafficking and sequestration of thousands of different RNA molecules to distinct cellular locations. Misregulation of this process leads to detrimental phenotypes in a wide range of systems, from developmental defects in Drosophila to neurological disease in humans. Despite this importance, our knowledge of the regulation of RNA localization is quite limited. For other modes of post-transcriptional regulation like splicing, our understanding of how the interactions of RNA binding proteins (RBPs) and RNA motifs lead to specific outcomes is much more mature. This relies on many years of work by many groups that have defined the regulatory language of splicing and allows us to make predictive and combinatorial models about how splicing is regulated across conditions and cellular environments. We lack such an ability with regards to RNA localization, in large part because we lack the analogous “parts list” that defines the language of localization regulation. Generally, the effect of RBP/RNA binding on post-transcriptional regulatory processes like splicing or RNA decay is consistent across cell types. For example, if an RBP promotes the splicing of an exon in one cell type, it often exerts a similar effect on that exon in another cell type. However, because RNA localization is inherently tied to cell morphology, the generality of localization regulation across cell types is unknown. Combinations of RNA motifs and RBPs that result in RNA localization to projections in neurons are also broadly present in non-neuronal cell types. Are these RNAs trafficked in non-neuronal cells? If so, to where? The answers to these questions first require a better knowledge of the underlying regulatory language of localization. The experiments proposed here are the beginnings of our efforts to define this language and test its generality. We have developed methods to isolate and profile subcellular transcriptomes from the projections of neurons and the apical and basal regions of epithelial cells. We will use these techniques to take a biochemical and transcriptome-wide approach to defining RBP/RNA interactions that regulate localization in two mammalian cell types: neurons and intestinal epithelial cells. By identifying transcripts that are mislocalized in RBP-null cells, we will identify functional RBP/RNA interactions. Using a massively parallel reporter assay, we will take an unbiased approach to finding RNA sequences that regulate localization. By comparing the activities of identified functional RBP/RNA interactions across cell types, we will for the first time be able to directly assess the generality of RNA localization. This methodical and innovative approach is the first step in our efforts to shed light on this fundamental but poorly understood cellular process.

概括 真核细胞内含有无数空间上不同的位点，这些位点具有多种功能。 为了促进这种组织，真核基因表达通常通过运输进行空间调节 将数千种不同的 RNA 分子隔离到不同的细胞位置。对此的监管不当 该过程会导致多种系统中的有害表型，例如果蝇的发育缺陷 导致人类神经系统疾病。 尽管如此重要，我们对 RNA 定位调控的了解却相当有限。对于其他 剪接等转录后调控模式，我们对 RNA 结合如何相互作用的理解 导致特定结果的蛋白质 (RBP) 和 RNA 基序更加成熟。这依赖于多年 许多小组的工作已经定义了剪接的监管语言，并使我们能够做出预测 以及关于如何在条件和细胞环境中调节剪接的组合模型。我们 缺乏这种RNA定位的能力，很大程度上是因为我们缺乏类似的“零件清单” 定义本地化法规的语言。 一般来说，RBP/RNA 结合对转录后调控过程（如剪接或转录）的影响 RNA 衰减在不同细胞类型中是一致的。例如，如果 RBP 促进一个细胞中外显子的剪接 类型，它通常对另一种细胞类型中的外显子产生类似的影响。然而，由于 RNA 定位 由于与细胞形态存在内在联系，跨细胞类型的定位调节的普遍性尚不清楚。 RNA 基序和 RBP 的组合也导致 RNA 定位到神经元的投射 广泛存在于非神经元细胞类型中。这些 RNA 是否在非神经元细胞中运输？如果是的话，去哪里？ 这些问题的答案首先需要更好地了解底层监管语言 的本地化。这里提出的实验是我们努力定义这种语言和 测试其通用性。我们开发了从细胞中分离和分析亚细胞转录组的方法 神经元的投影以及上皮细胞的顶端和基底区域。我们将使用这些技术来采取 一种生化和转录组范围的方法来定义调节定位的 RBP/RNA 相互作用 两种哺乳动物细胞类型：神经元和肠上皮细胞。通过识别错误定位的转录本 在 RBP 缺失的细胞中，我们将鉴定功能性 RBP/RNA 相互作用。使用大规模并行报告分析， 我们将采取公正的方法来寻找调节定位的RNA序列。通过比较 确定跨细胞类型的功能性 RBP/RNA 相互作用的活性，我们将第一次能够 直接评估 RNA 定位的一般性。这种有条理和创新的方法是迈出的第一步 我们努力阐明这一基本但知之甚少的细胞过程。