Xiong Jun, Feng Tian, Yuan Bi-Feng
School of Public Health, Wuhan University, Wuhan 430071, China.
Se Pu. 2024 Jul;42(7):632-645. doi: 10.3724/SP.J.1123.2023.12025.
Over 170 chemical modifications have been discovered in various types of ribonucleic acids (RNAs), including messenger RNA (mRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA). These RNA modifications play crucial roles in a wide range of biological processes such as gene expression regulation, RNA stability maintenance, and protein translation. RNA modifications represent a new dimension of gene expression regulation known as the "epitranscriptome". The discovery of RNA modifications and the relevant writers, erasers, and readers provides an important basis for studies on the dynamic regulation and physiological functions of RNA modifications. Owing to the development of detection technologies for RNA modifications, studies on RNA epitranscriptomes have progressed to the single-base resolution, multilayer, and full-coverage stage. Transcriptome-wide methods help discover new RNA modification sites and are of great importance for elucidating the molecular regulatory mechanisms of epitranscriptomics, exploring the disease associations of RNA modifications, and understanding their clinical applications. The existing RNA modification sequencing technologies can be categorized according to the pretreatment approach and sequencing principle as direct high-throughput sequencing, antibody-enrichment sequencing, enzyme-assisted sequencing, chemical labeling-assisted sequencing, metabolic labeling sequencing, and nanopore sequencing technologies. These methods, as well as studies on the functions of RNA modifications, have greatly expanded our understanding of epitranscriptomics. In this review, we summarize the recent progress in RNA modification detection technologies, focusing on the basic principles, advantages, and limitations of different methods. Direct high-throughput sequencing methods do not require complex RNA pretreatment and allow for the mapping of RNA modifications using conventional RNA sequencing methods. However, only a few RNA modifications can be analyzed by high-throughput sequencing. Antibody enrichment followed by high-throughput sequencing has emerged as a crucial approach for mapping RNA modifications, significantly advancing the understanding of RNA modifications and their regulatory functions in different species. However, the resolution of antibody-enrichment sequencing is limited to approximately 100-200 bp. Although chemical crosslinking techniques can achieve single-base resolution, these methods are often complex, and the specificity of the antibodies used in these methods has raised concerns. In particular, the issue of off-target binding by the antibodies requires urgent attention. Enzyme-assisted sequencing has improved the accuracy of the localization analysis of RNA modifications and enables stoichiometric detection with single-base resolution. However, the enzymes used in this technique show poor reactivity, specificity, and sequence preference. Chemical labeling sequencing has become a widely used approach for profiling RNA modifications, particularly by altering reverse transcription (RT) signatures such as RT stops, misincorporations, and deletions. Chemical-assisted sequencing provides a sequence-independent RNA modification detection strategy that enables the localization of multiple RNA modifications. Additionally, when combined with the biotin-streptavidin affinity method, low-abundance RNA modifications can be enriched and detected. Nevertheless, the specificity of many chemical reactions remains problematic, and the development of specific reaction probes for particular modifications should continue in the future to achieve the precise localization of RNA modifications. As an indirect localization method, metabolic labeling sequencing specifically localizes the sites at which modifying enzymes act, which is of great significance in the study of RNA modification functions. However, this method is limited by the intracellular labeling of RNA and cannot be applied to biological samples such as clinical tissues and blood samples. Nanopore sequencing is a direct RNA-sequencing method that does not require RT or the polymerase chain reaction (PCR). However, challenges in analyzing the data obtained from nanopore sequencing, such as the high rate of false positives, must be resolved. Discussing sequencing analysis methods for various types of RNA modifications is instructive for the future development of novel RNA modification mapping technologies, and will aid studies on the functions of RNA modifications across the entire transcriptome.
在各种类型的核糖核酸（RNA）中已发现超过170种化学修饰，包括信使RNA（mRNA）、核糖体RNA（rRNA）、转运RNA（tRNA）和小核RNA（snRNA）。这些RNA修饰在广泛的生物学过程中发挥着关键作用，如基因表达调控、RNA稳定性维持和蛋白质翻译。RNA修饰代表了基因表达调控的一个新维度，即“表观转录组”。RNA修饰以及相关的写入酶、擦除酶和读取蛋白的发现，为研究RNA修饰的动态调控和生理功能提供了重要基础。由于RNA修饰检测技术的发展，RNA表观转录组学研究已进入单碱基分辨率、多层次和全基因组覆盖阶段。全转录组方法有助于发现新的RNA修饰位点，对于阐明表观转录组学的分子调控机制、探索RNA修饰与疾病的关联以及理解其临床应用具有重要意义。现有的RNA修饰测序技术可根据预处理方法和测序原理分为直接高通量测序、抗体富集测序、酶辅助测序、化学标记辅助测序、代谢标记测序和纳米孔测序技术。这些方法以及对RNA修饰功能的研究极大地扩展了我们对表观转录组学的理解。在本综述中，我们总结了RNA修饰检测技术的最新进展，重点介绍了不同方法的基本原理、优点和局限性。直接高通量测序方法不需要复杂的RNA预处理，可使用传统RNA测序方法对RNA修饰进行定位。然而，高通量测序只能分析少数几种RNA修饰。抗体富集后进行高通量测序已成为定位RNA修饰的关键方法，显著推动了对不同物种中RNA修饰及其调控功能的理解。然而，抗体富集测序的分辨率限制在约100 - 200 bp。虽然化学交联技术可以实现单碱基分辨率，但这些方法通常很复杂，并且这些方法中使用的抗体的特异性也受到关注。特别是，抗体的脱靶结合问题需要紧急关注。酶辅助测序提高了RNA修饰定位分析的准确性，并能够以单碱基分辨率进行化学计量检测。然而，该技术中使用的酶表现出较差的反应性、特异性和序列偏好性。化学标记测序已成为一种广泛用于分析RNA修饰的方法，特别是通过改变逆转录（RT）特征，如RT终止、错掺入和缺失。化学辅助测序提供了一种不依赖序列的RNA修饰检测策略，能够对多种RNA修饰进行定位。此外，当与生物素 - 链霉亲和素亲和方法结合时，可以富集和检测低丰度RNA修饰。然而，许多化学反应的特异性仍然存在问题，未来应继续开发针对特定修饰的特异性反应探针，以实现RNA修饰的精确定位。作为一种间接定位方法，代谢标记测序专门定位修饰酶作用的位点，这在RNA修饰功能研究中具有重要意义。然而，该方法受到RNA细胞内标记的限制，不能应用于临床组织和血液样本等生物样本。纳米孔测序是一种直接RNA测序方法，不需要RT或聚合酶链反应（PCR）。然而，必须解决纳米孔测序数据分析中的挑战，如高假阳性率。讨论各种类型RNA修饰的测序分析方法对新型RNA修饰定位技术的未来发展具有指导意义，并将有助于对整个转录组中RNA修饰功能的研究。
