Harnessing Nature's Molecular Recognition Capabilities to Map and Study RNA Modifications.
Harnessing Nature's Molecular Recognition Capabilities to Map and Study RNA Modifications.
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利用自然界的分子识别能力来绘制和研究RNA修饰。
DOI：
10.1021/acs.accounts.2c00287
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发表时间：
2022-08-16
影响因子：
18.3
通讯作者：
Heemstra, Jennifer M.
中科院分区：
文献类型：
--
作者：
Felix, Ansley S.;Quillin, Alexandria L.;Mousavi, Shikufa;Heemstra, Jennifer M.
RNA编辑或“表观转录组修饰”是指转录后发生的RNA加工，以改变核酸的序列或结构。这些化学改变可以在核糖或核碱基上发现，虽然许多化学改变是“沉默的”并且不会改变RNA的沃森-克里克-富兰克林密码，但其他化学改变会导致重新编码事件。迄今为止，已鉴定出 170 多种 RNA 修饰，每种修饰都有特定的生物学目的。此外，RNA 编辑失调与多种疾病和失调有关。随着新修饰的发现以及我们对其功能影响的了解不断加深，对识别和绘制转录组中编辑位点的选择性方法的需求也随之增加。研究 RNA 修饰的最常见方法依赖于抗体作为亲和试剂；然而，抗体可能难以生成，并且常常具有不良的脱靶结合。最近，选择性化学标记通过提供可用于检测、富集和定量 RNA 修饰的技术，推动了该领域的发展。在我们使用丙烯酰胺进行肌苷标记的方法中，我们证明了这种方法能够使用其他标签或亲和手柄进行下拉或下游功能化。虽然这种方法确实能够对 A 到 I 编辑水平进行定量分析，但我们发现选择性存在显着的限制，这可能是由于肌苷和假尿苷或其他核碱基的相似反应性特征。为了克服抗体和化学标记方法的固有局限性，研究表观转录组的最新方法是重新利用识别修饰 RNA 的蛋白质和酶。我们的实验室使用了核酸内切酶 V（一种修复酶，可以切割含有肌苷的 RNA），并将其重新编程为结合肌苷。我们首先利用 EndoV 开发一种 RNA 测序制备技术，我们称之为 EndoVIPER-seq。该方法使用 EndoV 来富集肌苷编辑的 RNA，从而在 RNA 测序中提供更好的覆盖范围，并导致以前未检测到的 A 到 I 编辑位点的发现。我们还利用 EndoV 创建了一种基于板的免疫测定 (EndoVLISA) 来量化细胞 RNA 中的肌苷。这种方法可以检测跨组织类型或疾病状态的差异 A 到 I 编辑水平，同时独立于 RNA 测序，使其具有成本效益和高通量。通过利用这种酶的分子识别能力，我们证明 EndoV 可以重新用作“抗肌苷抗体”，以开发从细胞 RNA 中检测和富集肌苷的新方法。大自然已经进化出大量能够选择性识别并作用于 RNA 修饰的蛋白质和酶，利用这些生物分子的亲和力为表观转录组学领域提供了一个有希望的新方向。
RNA editing or “epitranscriptomic modification” refers to processing of RNA that occurs after transcription to alter the sequence or structure of the nucleic acid. These chemical alterations can be found on either the ribose sugar or the nucleobase, and while many are “silent” and do not change the Watson-Crick-Franklin code of the RNA, others result in recoding events. Over 170 RNA modifications have been identified so far, each having a specific biological purpose. Additionally, dysregulated RNA editing has been linked to several types of diseases and disorders. As new modifications are discovered and our understanding of their functional impact grows, so does the need for selective methods of identifying and mapping editing sites in the transcriptome. The most common methods for studying RNA modifications rely on antibodies as affinity reagents; however, antibodies can be difficult to generate and often have undesirable off-target binding. More recently, selective chemical labeling has advanced the field by offering techniques that can be used for detection, enrichment, and quantification of RNA modifications. In our method using acrylamide for inosine labeling, we demonstrated the versatility with which this approach enables pull-down or downstream functionalization with other tags or affinity handles. While this method did enable quantitative analysis of A-to-I editing levels, we found that selectivity posed a significant limitation, likely due to the similar reactivity profiles of inosine and pseudouridine or other nucleobases. Seeking to overcome the inherent limitations of antibodies and chemical labeling methods, a more recent approach to studying the epitranscriptome is through repurposing of proteins and enzymes that recognize modified RNA. Our lab has used Endonuclease V, a repair enzyme that cleaves inosine-containing RNAs, and reprogrammed it to instead bind inosine. We first harnessed EndoV to develop a preparative technique for RNA sequencing that we termed EndoVIPER-seq. This method uses EndoV to enrich inosine-edited RNAs, providing better coverage in RNA sequencing and leading to the discovery of previously undetected A-to-I editing sites. We also leveraged EndoV to create a plate-based immunoassay (EndoVLISA) to quantify inosine in cellular RNA. This approach can detect differential A-to-I editing levels across tissue types or disease states while being independent of RNA sequencing, making it cost-effective and high-throughput. By harnessing the molecular recognition capabilities of this enzyme, we show that EndoV can be repurposed as an “anti-inosine antibody” to develop new methods of detecting and enriching inosine from cellular RNA. Nature has evolved a plethora of proteins and enzymes that selectively recognize and act on RNA modifications, and exploiting the affinity of these biomolecules offers a promising new direction for the field of epitranscriptomics.
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RNA 假尿苷化：对旧修饰的新见解。
影响因子：
13.8
作者：
Ge, Junhui;Yu, Yi-Tao
通讯作者：
Yu, Yi-Tao
DOI：
10.1007/978-1-4939-2253-6_14
发表时间：
2015-01-01
期刊：
NUCLEAR BODIES AND NONCODING RNAS: METHODS AND PROTOCOLS
影响因子：
--
作者：
Cozzitorto, Joseph A.;Jimbo, Masaya;Brody, Jonathan R.
通讯作者：
Brody, Jonathan R.
影响因子：
4.1
作者：
Feederle R;Schepers A
通讯作者：
Schepers A
影响因子：
15
作者：
Himo, F;Lovell, T;Fokin, VV
通讯作者：
Fokin, VV
影响因子：
6.2
作者：
Chowdhury B;Cho IH;Hahn N;Irudayaraj J
通讯作者：
Irudayaraj J