Understanding the regulatory language of RNA localization

了解 RNA 定位的调控语言

• 批准号: 10663827
• 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/10663827
• 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; Genome engineering; Human; Knowledge; Language; Lead; 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; Testing; Time; Transcript; Untranslated RNA; Work; cell type; combinatorial; experimental study; high throughput screening; innovation; intestinal epithelium; nervous system disorder; next generation sequencing; posttranscriptional; 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.

总结 真核细胞内含有无数空间上不同的位点，这些位点发挥各种功能。 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控。 以及将数千种不同的RNA分子隔离到不同的细胞位置。这是一个错误的调节 这一过程导致在广泛的系统中有害的表型，从果蝇的发育缺陷， 人类神经系统疾病。 尽管如此重要，我们对RNA定位调控的了解还是相当有限的。为其他 转录后调节的模式，如剪接，我们对RNA结合的相互作用的理解 蛋白质（RBP）和RNA基序导致特定结果的方法要成熟得多。这依赖于多年来 许多小组的工作已经定义了剪接的调控语言，并使我们能够预测 以及关于剪接如何在各种条件和细胞环境中调节的组合模型。我们 在RNA定位方面缺乏这样的能力，很大程度上是因为我们缺乏类似的“部件列表”， 定义本地化法规的语言。 通常，RBP/RNA结合对转录后调节过程如剪接或转录后调节过程的影响是不确定的。 RNA衰变在不同的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， 在一种细胞类型中，它通常对另一种细胞类型中的该外显子产生类似的影响。然而，由于RNA定位是 固有地与细胞形态相联系，跨细胞类型的定位调节的一般性是未知的。 RNA基序和RBP的组合导致RNA定位于神经元中的投射， 广泛存在于非神经元细胞类型中。这些RNA在非神经元细胞中被运输吗？如果是，去哪里？ 这些问题的答案首先需要更好地了解基本的监管语言 本地化。这里提出的实验是我们努力定义这种语言的开始， 测试其通用性。我们已经开发了从细胞中分离和分析亚细胞转录组的方法。 神经元的投射以及上皮细胞的顶端和基底区域。我们将利用这些技术 一种生物化学和转录组范围的方法来定义RBP/RNA相互作用，调节定位在 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缺失细胞中，我们将鉴定功能性RBP/RNA相互作用。使用大规模平行报道分析， 我们将采取无偏见的方法来寻找调节定位的RNA序列。通过比较 通过对不同细胞类型的RBP/RNA相互作用的研究，我们将首次能够 直接评估RNA定位的一般性。这种有条不紊的创新方法是 我们努力揭示这一基本但知之甚少的细胞过程。