STAMP technology to enable single-cell and isoform-sensitive detection of RBP sites

STAMP 技术可实现 RBP 位点的单细胞和亚型敏感检测

• 批准号: 10277360
• 负责人: Eugene Wei-Ming Yeo
• 金额: $ 99.54万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2025-05-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10277360
• 关键词: Adoption; Amino Acids; Antibodies; Binding; Binding Proteins; Binding Sites; Biological; Birth; Cells; Collection; Communities; Coupled; Data; Data Set; Detection; Disease; Elements; Gene Expression; Genetic Translation; Genomics; Goals; Grant; Human; Immunoprecipitation; Label; Libraries; Life Cycle Stages; Locales; Measurement; Measures; Mediating; Messenger RNA; Metabolism; Methodology; Methods; Modification; Molecular; Monitor; Morphologic artifacts; National Human Genome Research Institute; Nature; Noise; Protein Isoforms; Protein Subunits; Protocols documentation; RNA; RNA Processing; RNA analysis; RNA-Binding Proteins; Reagent; Regulatory Element; Reproducibility; Research; Resolution; Resources; Ribosomal Proteins; Ribosomes; Role; Sampling; Signal Transduction; Site; Specificity; Structure; Surveys; Technology; Testing; Third Generation Sequencing; Time; Transcript; Translations; Untranslated RNA; Work; analytical tool; apoB mRNA editing catalytic subunit; base; cell type; computer framework; crosslink; design; detection limit; flexibility; genomic RNA; improved; insight; nanopore; new technology; novel; paralogous gene; programs; response; ribosome profiling; single cell analysis; single molecule; technological innovation; technology development; transcriptome; transcriptome sequencing

PROJECT SUMMARY RNA-binding proteins (RBPs) interact with RNA molecules from synthesis to decay to control their metabolism, subcellular localization, stability and translation. Methods for transcriptome-wide detection of RBP-RNA interactions provide insights into how RBPs regulate gene expression programs and how RNA processing is disrupted in disease state. Despite their association with disease and although the importance of regulating gene expression is well appreciated, only a small fraction of the over 2,000 RBPs identified thus far have known RNA targets and molecular roles. Commonly, immunoprecipitation-based technologies coupled to high throughput (Illumina) sequencing, such as RNA immunoprecipitation (RIP) and Crosslinking Immunoprecipitation (CLIP), and ribosome profiling are used to identify RBP targets and binding sites across the transcriptome. However, these experimental protocols are labor-intensive, require large amounts of input material, are not adaptable to high-throughput workflows. To overcome these limitations, we develop a novel technology, reagent resource, experimental protocols and a computational framework, that we collectively term STAMP (Surveying Targets By APOBEC-Mediated Profiling), for detecting RBP-RNA targets and translation at the single-cell and single- molecule level. In preliminary data we demonstrate, for the first time in the field, discovery of RBP-RNA sites and translation states at single-cell resolution. We anticipate that STAMP can be used reliably to identify RNA targets, binding sites and even extract motifs from a few cells to a single cell, thus effectively increasing limits of detection over current methods by several orders of magnitude. Combined with simultaneous RNA-seq analyses, STAMP will enable the combined identification of RBP binding sites and global measurement of gene expression, a long- standing goal for the gene expression, genomics and RNA communities. As a corollary, even without single cell analyses, STAMP can accept ultra-low input material which enables rare cell-types to be collected and analyzed for RBP-interactomes. By applying STAMP to ribosomal proteins, we extend this approach for single-cell detection of ribosome association while simultaneously measuring gene expression. Our conceptual and technological innovations will, for the first time, enable translation efficiency and RBP-interactomes to be measured at single-cell level and at scale, opening up new paradigms of biological questions.

项目总结 RNA结合蛋白(RBP)从合成到衰变与RNA分子相互作用，控制其代谢， 亚细胞定位、稳定性和翻译。RBP-RNA的全转录组检测方法 交互作用提供了对限制性商业惯例如何调节基因表达程序以及RNA处理是如何进行的深入了解 处于疾病状态的中断的。尽管它们与疾病有关，尽管调控基因的重要性 表达是很好的理解，到目前为止发现的2000多个限制性商业惯例中只有一小部分是已知的RNA 靶标和分子作用。通常，基于免疫沉淀的技术与高吞吐量相结合 (Illumina)测序，如RNA免疫沉淀(RIP)和交联免疫沉淀(CLIP)， 和核糖体图谱用于识别转录组中的RBP靶标和结合位点。然而， 这些实验方案是劳动密集型的，需要大量的输入材料，不适合 高吞吐量工作流。为了克服这些限制，我们开发了一种新技术，试剂资源， 实验协议和计算框架，我们统称为STAMP(通过以下方式测量目标 APOBEC介导的轮廓分析)，用于检测RBP-RNA靶标并在单细胞和单细胞- 分子水平。在初步数据中，我们首次在该领域证明了RBP-RNA位点和 单单元分辨率的平移状态。我们预计该印记可以可靠地用于识别RNA靶标， 结合位点，甚至从几个细胞中提取基序到单个细胞，从而有效地提高了检测限 比目前的方法高出几个数量级。与同时的RNA-SEQ分析相结合，STAMP 将使RBP结合位点的鉴定和基因表达的全球测量成为可能，一个长期的- 基因表达、基因组学和RNA社区的长期目标。作为必然结果，即使没有单个细胞 分析，STAMP可以接受超低输入材料，使稀有细胞类型能够被收集和分析 对于RBP-Interacomes。通过将STAMP应用于核糖体蛋白质，我们将该方法扩展到单细胞 检测核糖体结合，同时测量基因表达。我们的概念和 技术创新将首次使翻译效率和RBP互动成为可能 在单细胞水平和规模上进行测量，开辟了生物学问题的新范式。