PDF(Pubmed)
Abstract：
RNA editing or \"epitranscriptomic modification\" refers to the 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 although many are \"silent\" and do not change the Watson-Crick-Franklin code of the RNA, others result in recoding events. More than 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 the 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. Although this method did enable the quantitative analysis of A-to-I editing levels, we found that selectivity posed a significant limitation, likely because of 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 the repurposing of proteins and enzymes that recognize modified RNA. Our laboratory 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.
摘要：
RNA编辑或“表位基因组修饰”是指在转录后发生的RNA加工以改变核酸的序列或结构。这些化学变化可以在核糖或核碱基上发现，尽管许多人“沉默”并且不改变RNA的沃森-克里克-富兰克林密码，其他人导致重新编码事件。到目前为止，已经确定了170多个RNA修饰。每个都有特定的生物学目的。此外,RNA编辑失调与几种类型的疾病和病症有关。随着新修改的发现和我们对其功能影响的理解的增长，在转录组中识别和定位编辑位点的选择性方法的需求也是如此。研究RNA修饰的最常见方法依赖于抗体作为亲和试剂；然而，抗体可能难以产生并且通常具有不期望的脱靶结合。最近,选择性化学标记通过提供可用于检测的技术，富集,和RNA修饰的定量。在我们使用丙烯酰胺标记肌苷的方法中，我们证明了这种方法能够实现与其他标签或亲和句柄的下拉或下游功能化的多功能性。尽管这种方法确实可以对A到I编辑水平进行定量分析，我们发现选择性造成了很大的限制，可能是因为肌苷和假尿苷或其他核碱基的相似反应性。寻求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通过识别修饰RNA的蛋白质和酶的再利用。我们的实验室已经使用了核酸内切酶V,一种能切割含肌苷的RNA的修复酶，并将其重新编程为结合肌苷。我们首先利用EndoV开发了一种用于RNA测序的制备技术，我们将其称为EndoVIPER-seq。这种方法使用EndoV来富集肌苷编辑的RNA，在RNA测序中提供更好的覆盖，并导致发现以前未检测到的A到I编辑位点。我们还利用EndoV来创建基于平板的免疫测定(EndoVLISA)来定量细胞RNA中的肌苷。这种方法可以检测组织类型或疾病状态之间的差异A到I编辑水平，而不依赖于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可以被重新用作“抗肌苷抗体”，以开发从细胞RNA中检测和富集肌苷的新方法。自然界已经进化出大量的蛋白质和酶，它们选择性地识别和作用于RNA修饰，利用这些生物分子的亲和力为表观组学领域提供了一个有前途的新方向。
