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: 10632150
• 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/10632150
• 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; Genomics; Goals; Grant; Human; Immunoprecipitation; Label; Libraries; Life Cycle Stages; Ligation; Locales; Maps; 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-Binding Proteins; RNA-Protein Interaction; Reagent; Regulatory Element; Reproducibility; Research; Resolution; Resources; Ribosomal Proteins; Ribosomes; Role; Sampling; Signal Transduction; Site; Specificity; Surveys; Technology; Testing; Third Generation Sequencing; Time; Transcript; Translations; Untranslated RNA; Work; analytical tool; apoB mRNA editing catalytic subunit; cell type; computer framework; crosslink; design; detection limit; flexibility; improved; insight; mRNA Translation; 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.

项目摘要 RNA结合蛋白（RBP）与从合成到衰减的RNA分子相互作用，以控制其代谢， 亚细胞定位，稳定性和翻译。 RBP-RNA转录组检测的方法 相互作用提供了有关RBP调节基因表达程序以及RNA处理的见解 处于疾病状态。尽管它们与疾病相关，尽管调节基因的重要性 表达得到很好的理解，到目前为止鉴定出的2,000多个RBP中，只有一小部分已知RNA 目标和分子角色。通常，基于免疫沉淀的技术与高通量结合 （Illumina）测序，例如RNA免疫沉淀（RIP）和交联的免疫沉淀（夹）， 核糖体分析用于识别RBP靶标和跨转录组的结合位点。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 高通量工作流程。为了克服这些局限性，我们开发了一种新颖的技术，试剂资源， 实验协议和计算框架，我们共同列为邮票（通过 APOBEC介导的分析），用于检测RBP-RNA靶标和单细胞和单细胞的翻译 分子水平。在初步数据中，我们在现场首次证明了RBP-RNA位点和 单细胞分辨率的翻译状态。我们预计可以可靠地使用邮票来识别RNA目标， 结合位点，甚至从几个细胞中提取基序到一个单元，从而有效地增加了检测的极限 通过几个数量级，在当前方法上。与同时的RNA-Seq分析结合在一起，邮票 将实现RBP结合位点的综合鉴定和基因表达的全球测量，这是长期的 基因表达，基因组学和RNA社区的常规目标。作为推论，即使没有单细胞 分析，邮票可以接受超低输入材料，从而可以收集和分析稀有的细胞类型 用于RBP - 相关。通过将邮票应用于核糖体蛋白，我们扩展了单细胞的方法 核糖体关联的检测，同时测量基因表达。我们的概念和 技术创新将首次实现翻译效率，而RBP Interactomes成为 以单细胞水平和大规模测量，开辟了生物学问题的新范式。