High-throughput dissection of RNA localization regulatory elements

RNA 定位调控元件的高通量解析

• 批准号: 10749328
• 负责人: Charlie E Moffatt
• 金额: $ 3.64万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10749328
• 关键词: 3' Untranslated Regions; Ablation; Acylation; Automobile Driving; Bioinformatics; Biological Assay; Biological Process; Cell Fractionation; Chemicals; Code; Data; Disease; Dissection; Elements; Embryonic Development; Functional disorder; GDF11 gene; High-Throughput Nucleotide Sequencing; Hydroxyl Radical; Knowledge; Language; Length; Location; Maps; Measures; Mediating; Molecular; Mutation; Nature; Neurites; Neurons; Nucleotides; Oligonucleotides; Physiological; Primer Extension; Process; RNA; RNA Sequences; RNA Transport; RNA-Binding Proteins; Regulatory Element; Reporter; Slide; Structure; Techniques; Testing; Transcript; Transgenes; Translational Regulation; Work; base; design; mutant; trafficking

PROJECT SUMMARY/ ABSTRACT RNA localization is critical for a diverse set of biological processes. The localization of an RNA depends on cis-elements, features inherent to the transcript, and trans-factors, effectors independent of the target transcript, which are often RNA binding proteins. Cis-elements that regulate RNA localization, often called "zip-codes," are often found in the 3′ untranslated regions (UTRs) of transcripts. However, for the more than 99% of the known localized RNAs, the cis-elements that regulate their localization are unknown. Recent work from the Taliaferro lab identified several 260 nucleotide RNA sequences within the 3’ UTRs of some neurite-localized RNAs that were necessary and sufficient for neurite RNA transport. These were identified using a massively parallel reporter assay that screened approximately 10,000 RNA sequences drawn from endogenous 3’ UTRs for their ability to traffick a reporter transcript to neurites. Interestingly, 100 nt subsequences of these 260 nt active elements were not capable of directing RNA transport. This indicates that (1) the minimal regulatory elements are quite large (likely longer than 100 nt) and (2) the true character of the localization regulatory elements remains unknown. In this work, it is proposed to comprehensively characterize the previously identified RNA localization regulatory elements and zero in on their important features. This will be done by generating a pool of 10,000 RNA sequences based on the previously identified 260 nt zipcodes. Each sequence in this pool will contain defined deletions of varying sizes that span the length of the zipcode. By integrating these mutants into the 3’ UTR of a reporter transcript assaying which of them retain the ability to direct localization of the reporter to neurites, a quantitative readout of the functional importance of each nucleotide within the 260 nt zipcode will be obtained. From this, a clear picture of the important features that make up active localization elements will arise, facilitating their identification in other localized RNAs. The large size of these zipcodes suggests that their secondary may be important for their activity. To test this, their secondary structure will be determined using chemical probing techniques. To test the functionality of the structure, thousands of mutants that disrupt RNA structure as well as compensatory mutants that restore it will be generated. As above, the ability of each of these mutants to drive a reporter transcript to neurites will be tested. In this way, RNA structure and function will be directly related. Answering these questions will help in understanding the underlying mechanisms of RNA localization as very few examples of RNA localization have known mechanistic underpinnings. Identifying the mechanism of RNA localization under physiological conditions is important to being able to understand potential dysfunction in disease states.

项目概要/摘要 RNA 定位对于多种生物过程至关重要。 顺式元件，转录本固有的特征，反式因子，独立于靶标的效应子 转录物，通常是调节 RNA 定位的顺式元件，通常称为 RNA 结合蛋白。 “邮政编码”通常出现在转录本的 3' 非翻译区 (UTR)，但也不止如此。 99% 的已知定位 RNA，调节其定位的顺式元件是未知的。 来自 Taliaferro 实验室的研究人员在一些基因的 3' UTR 内鉴定出了几个 260 个核苷酸的 RNA 序列。 神经突定位的 RNA 对于神经突 RNA 运输是必要且充分的。 使用大规模并行检测报告仪筛选了大约 10,000 个 RNA 序列 内源性 3’ UTR 能够将报告基因转录物运输到神经突。 这些 260 nt 活性元件的子序列不能指导 RNA 转运。 (1) 最小调控元件相当大（可能长于 100 nt）并且 (2) 的真实特征 本地化监管要素仍然未知。 在这项工作中，建议全面表征先前鉴定的 RNA 定位 监管要素并将其重要特征归零 这将通过生成 10,000 个池来完成。 基于先前识别的 260 nt 邮政编码的 RNA 序列此池中的每个序列都将包含。 通过将这些突变体整合到 3' 区，定义了跨越邮政编码长度的不同大小的删除。 报告基因转录物测定的 UTR，其中保留了直接定位报告基因的能力 神经突，260 nt 邮政编码内每个核苷酸功能重要性的定量读数将是 由此，我们将清楚地了解构成主动本地化元素的重要特征。 出现，促进它们在其他局部 RNA 中的识别。 这些邮政编码的大尺寸表明它们的辅助可能对其活动很重要为了测试这一点。 它们的二级结构将使用化学探测技术来确定，以测试其功能。 结构，破坏 RNA 结构的数千个突变体以及恢复它的补偿突变体将 如上所述，将生成这些突变体中的每一个将报告基因转录物驱动至神经突的能力。 这样一来，RNA的结构和功能就会直接相关。 回答这些问题将有助于理解 RNA 定位的根本机制 很少有 RNA 定位的例子具有已知的机制基础。 生理条件下的定位对于能够理解潜在的功能障碍非常重要 疾病状态。