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.

项目摘要/摘要 RNA定位对于各种生物过程至关重要。 RNA的定位取决于 顺式元素，继承为成绩单的特征和跨因素，效果与目标无关 转录本通常是RNA结合蛋白。调节RNA定位的顺式元素，通常称为 经常在转录本的3'未翻译区域（UTR）中发现“拉链代码”。但是，超过 99％的已知局部RNA，调节其定位的顺式元素是未知的。最近的工作 从塔利亚弗罗实验室（Taliaferro Lab 神经定位的RNA对于神经RNA转运所必需而足够。这些被确定 使用大量平行的记者测定法，该测定法筛选了大约10,000个RNA序列 内源性3'UTRS的能力将记者转录物传递到神经上。有趣的是，100 nt 这260 NT活性元件的子序列无法指导RNA转运。这表明 （1）最小调节元素很大（可能长于100 nt），（2） 定位调节元素仍然未知。 在这项工作中，建议全面地表征先前确定的RNA定位 监管元素的重要特征为零。这将通过产生10,000的池来完成 基于先前鉴定的260 NT邮政编码的RNA序列。该池中的每个序列都将包含 跨越邮政编码长度的不同大小的定义缺失。通过将这些突变体集成到3' 记者成绩单分析的UTR，其中哪个保留了将记者定位到的能力 神经运动，对260 nt Zipcode中每个核苷酸的功能重要性的定量读数将是 获得。由此，清楚地了解了构成主动本地化元素的重要功能 出现，支持他们在其他局部RNA中的识别。 这些Zipcodes的大尺寸表明，其次要对其活动可能很重要。为了测试这一点， 它们的二级结构将使用化学探测技术确定。测试功能 结构，数千个破坏RNA结构的突变体以及恢复它的补偿性突变体 被生成。如上所述，这些突变体中每个突变体的能力将报告基因转录物驱动到神经影响 测试。这样，RNA结构和功能将直接相关。 回答这些问题将有助于理解RNA定位的潜在机制 RNA定位的例子很少有已知的机械基础。识别RNA的机制 生理条件下的定位对于能够了解潜在功能障碍很重要 疾病状态。