Zaringhalam Maryam, Papavasiliou F Nina
Laboratory of Lymphocyte Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, United States; The Rockefeller Graduate Program, The Rockefeller University, 1230 York Avenue, New York, NY 10065, United States.
Laboratory of Lymphocyte Biology, The Rockefeller University, 1230 York Avenue, New York, NY 10065, United States.
Methods. 2016 Sep 1;107:63-72. doi: 10.1016/j.ymeth.2016.03.001. Epub 2016 Mar 8.
The isomerization of uridine to pseudouridine (Ψ), known as pseudouridylation, is the most abundant post-transcriptional modification of stable RNAs. Due to technical limitations in pseudouridine detection methods, studies on pseudouridylation have historically focused on ribosomal RNAs, transfer RNAs, and spliceosomal small nuclear RNAs, where Ψs play a critical role in RNA biogenesis and function. Recently, however, a series of deep sequencing methods-Pseudo-seq, Ψ-seq, PSI-seq, and CeU-seq-has been published to map Ψ positions across the entire transcriptome with single nucleotide resolution. These data have greatly expanded the catalogue of pseudouridylated transcripts, which include messenger RNAs and noncoding RNAs. Furthermore, these methods have revealed conditionally-dependent sites of pseudouridylation that appear in response to cellular stress, suggesting that pseudouridylation may play a role in dynamically modulating RNA function. Collectively, these methods have opened the door to further study of the biological relevance of naturally occurring Ψs. However, an in-depth comparison of these techniques and their results has not yet been undertaken despite all four methods relying on the same basic principle: Ψ detection through selective chemical labeling by the carbodiimide known as CMC. In this article, we will outline the currently available high-throughput Ψ-detection methods and present a comparative analysis of their results. We will then discuss the merits and limitations of these approaches, including those inherent in CMC conjugation, and their potential to further elucidate the function of this ubiquitous and dynamic modification.
尿苷异构化为假尿苷（Ψ），即假尿苷化，是稳定RNA最丰富的转录后修饰。由于假尿苷检测方法存在技术局限性，历史上关于假尿苷化的研究主要集中在核糖体RNA、转运RNA和剪接体小核RNA上，其中假尿苷在RNA生物合成和功能中起着关键作用。然而，最近一系列深度测序方法——假尿苷测序（Pseudo-seq）、Ψ测序（Ψ-seq）、PSI测序（PSI-seq）和CeU测序（CeU-seq）——已被发表，能够以单核苷酸分辨率绘制整个转录组中的假尿苷位置。这些数据极大地扩展了假尿苷化转录本的目录，其中包括信使RNA和非编码RNA。此外，这些方法还揭示了响应细胞应激而出现的条件依赖性假尿苷化位点，这表明假尿苷化可能在动态调节RNA功能中发挥作用。总体而言，这些方法为进一步研究天然存在的假尿苷的生物学相关性打开了大门。然而，尽管这四种方法都基于相同的基本原理：通过被称为CMC的碳二亚胺进行选择性化学标记来检测假尿苷，但尚未对这些技术及其结果进行深入比较。在本文中，我们将概述目前可用的高通量假尿苷检测方法，并对其结果进行比较分析。然后，我们将讨论这些方法的优缺点，包括CMC缀合所固有的优缺点，以及它们进一步阐明这种普遍存在且动态的修饰功能的潜力。
