An ultra-low-input RNase footprinting assay to quantify cytosolic and mitochondrial translation simultaneously

超低输入 RNase 足迹分析可同时量化胞质和线粒体翻译

基本信息

批准号：
10551894
负责人：
Zhe Ji
金额：
$ 48万
依托单位：
NORTHWESTERN UNIVERSITY AT CHICAGO
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-01-17 至 2025-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10551894
关键词：
Address Amino Acids Biological Assay Blood Bone Marrow CRISPR library CRISPR screen Cell Count Cell Differentiation process Cells Codon Nucleotides Complex Consumption Cultured Cells Data Dependence Development Digestion Disease Progression Freezing Genes Genome Genomics Hematopoiesis Hematopoietic Hematopoietic stem cells Heterogeneity Hour Individual Knock-out Length Libraries Lymphocyte Malignant Neoplasms Maps Mass Spectrum Analysis Mediating Methods Mitochondria Molecular Normal Cell Nucleotides Open Reading Frames Oxidative Phosphorylation Peptides Physiological Preparation Procedures Protein Biosynthesis Protocols documentation RNA Regulation Research Resolution Ribonucleases Ribosomal RNA Ribosomes Role Sucrose Techniques Technology Time Tissue Sample Tissues Translating Translation Initiation Translational Regulation Translations Ultracentrifugation Work cancer cell cell growth cell type clinical prognosis cost design genome-wide human disease in vivo insight mitochondrial genome mitochondrial metabolism monocyte novel polysome profiling prevent ribosome profiling self-renewal stem cells tool transcriptome sequencing transcriptomics translation factor usability

Address Amino Acids Biological Assay Blood Bone Marrow CRISPR library CRISPR screen Cell Count Cell Differentiation process Cells Codon Nucleotides Complex Consumption Cultured Cells Data Dependence Development Digestion Disease Progression Freezing Genes Genome Genomics Hematopoiesis Hematopoietic Hematopoietic stem cells Heterogeneity Hour Individual Knock-out Length Libraries Lymphocyte Malignant Neoplasms Maps Mass Spectrum Analysis Mediating Methods Mitochondria Molecular Normal Cell Nucleotides Open Reading Frames Oxidative Phosphorylation Peptides Physiological Preparation Procedures Protein Biosynthesis Protocols documentation RNA Regulation Research Resolution Ribonucleases Ribosomal RNA Ribosomes Role Sucrose Techniques Technology Time Tissue Sample Tissues Translating Translation Initiation Translational Regulation Translations Ultracentrifugation Work cancer cell cell growth cell type clinical prognosis cost design genome-wide human disease in vivo insight mitochondrial genome mitochondrial metabolism monocyte novel polysome profiling prevent ribosome profiling self-renewal stem cells tool transcriptome sequencing transcriptomics translation factor usability

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项目摘要

ABSTRACT Ribosome profiling isolates ribosome-protected fragments for sequencing and reveals active translation at the single-nucleotide resolution in vivo. It represents a valuable approach to study various aspects of protein synthesis, such as the regulation of translation efficiency, alternative translation initiation, ribosome elongation and pausing, codon usage, and identifying non-canonical open reading frames and micropeptides (<100 amino acids) encoded in a genome. Thus, it provides unique molecule insights of translational control, which could not be achieved by other genomic technologies, such as mass spectrometry or polysome profiling. However, current ribosome profiling protocols generally use complicated experimental procedures to isolate ribosome-RNA complexes such as though a sucrose cushion, and require millions of input cells. This technical barrier has prevented its application to examing translation profiles of primary physiological tissue samples with low-input cell numbers. To tackle this long-standing challenge, here we propose to develop an ultra-low-input RNase footprinting approach for the rapid quantification of cytosolic and mitochondrial translation simultaneously. Our method simplified the experimental procedure to select ribosome footprints based on optimized RNase digestion. My lab has made the assay work well for as few as 1,000 cultured cells. In this proposal, we aim to further develop the assay and make it work robustly for a small amount of primary tissue samples (Aim 1). Furthermore, we will apply the method to map the RNA translation landscape of rare progenitor cells and differentiated cell types during hematopoiesis (Aim 2). The results will reveal novel mechanisms mediating the translational control underlying hematopoietic cell fate decisions. Finally, by leveraging that our assay provides a simplified method to study mitochondrial translation, we will use it to examine the heterogeneity of mitochondrial translation machinery and build the functional network mediated by individual factors (Aim 3). Altogether, the refined ultra- low-input RNase footprinting method developed in this proposal will be a valuable tool and can be widely used to study the translational regulation underlying complex physiological conditions from normal development to disease progression.

抽象的核糖体分析隔离核糖体保护的片段，用于测序，并揭示了在体内单核苷酸分辨率。它代表了研究蛋白质各个方面的宝贵方法合成，例如调节翻译效率，替代翻译起始，核糖体伸长率和暂停，密码子使用情况以及识别非典型的开放式阅读框和微肽（<100氨基酸）编码在基因组中。因此，它提供了转化控制的独特分子见解，这无法可以通过其他基因组技术来实现，例如质谱法或多谱分析。但是，当前核糖体分析方案通常使用复杂的实验程序来分离核糖体-RNA 诸如蔗糖垫子的复合物，需要数百万个输入细胞。这个技术障碍有阻止其应用于低输入的原代生理组织样品的翻译曲线单元格数。为了应对这一长期挑战，我们在这里建议开发超低输入的RNase 同时快速定量胞质和线粒体翻译的足迹方法。我们的方法简化了基于优化的RNase消化的实验过程，以选择核糖体足迹。我的实验室使得该测定法可以很好地工作，只有1,000个培养的细胞。在此提案中，我们的目标是进一步开发该测定法，使其可用于少量原代组织样品（AIM 1）。此外，我们将应用该方法来绘制稀有祖细胞和分化细胞的RNA翻译景观造血期间的类型（AIM 2）。结果将揭示介导翻译控制的新型机制潜在的造血细胞命运决定。最后，通过利用我们的测定法提供了一种简化的方法为了研究线粒体翻译，我们将使用它来检查线粒体翻译的异质性机械并构建由个别因素介导的功能网络（AIM 3）。总共，精制的超级本提案中开发的低输入RNase足迹方法将是一个有价值的工具，可以广泛使用研究从正常发育到疾病进展。