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.

项目总结/摘要 RNA定位对于一系列不同的生物过程至关重要。RNA的定位取决于 顺式元件，转录本固有的特征，和反式因子，独立于靶的效应物 转录物，通常是RNA结合蛋白。调节RNA定位的顺式元件，通常称为 “邮政编码”通常存在于转录本的3′非翻译区（UTR）中。然而，对于超过 99%的已知定位RNA，调节其定位的顺式元件是未知的。最近的工作 在一些哺乳动物的3'UTR中鉴定了几个260个核苷酸的RNA序列， 神经突定位的RNA是必要的和足够的神经突RNA运输。这些被确定为 使用大规模平行的报告基因分析，筛选了大约10，000个RNA序列， 内源性3'UTR，因为它们能够将报告转录物运送到神经突。有趣的是， 这些260 nt活性元件的序列不能指导RNA转运。这表明 (1)最小调节元件相当大（可能长于100 nt）和（2）最小调节元件的真正特征是（1）最小调节元件的最小调节元件的最小调节元件。 定位调节元件仍然未知。 在这项工作中，提出了全面表征先前确定的RNA定位 监管要素，并集中在其重要特征上。这将通过生成10，000个池来完成 基于先前鉴定的260 nt邮政编码的RNA序列。该池中的每个序列将包含 定义了跨越邮政编码长度的不同大小的删除。通过将这些突变体整合到3' 分析报告基因转录本的UTR，测定它们中的哪些保留了将报告基因直接定位于 神经突，260 nt邮政编码内每个核苷酸的功能重要性的定量读数将被 得到了由此，可以清楚地了解构成主动本地化元素的重要特性， 出现，促进它们在其他本地化RNA中的识别。 这些邮政编码的大尺寸表明，它们的次级可能对其活动很重要。为了验证这个， 它们的二级结构将使用化学探测技术来确定。要测试 结构，数以千计的突变体破坏RNA结构以及补偿突变体，恢复它将 生成。如上所述，这些突变体中的每一个将报告转录物驱动到神经突的能力将被削弱。 测试.这样，RNA的结构和功能将直接相关。 回答这些问题将有助于理解RNA定位的基本机制， RNA定位的几个例子具有已知的机制基础。识别RNA的机制 在生理条件下的定位对于能够理解潜在的功能障碍是重要的， 疾病状态。