紫外交联和蛋白免疫共沉淀技术的演进与创新
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作者单位:

1)内蒙古大学生命科学学院,呼和浩特 010021;2)复旦大学生命科学学院,上海 200438

作者简介:

Tel:那顺布和 0471-4996885,E-mail:bnashun@imu.edu.cnBuhe Nashun. Tel: 86-471-4996885,E-mail:bnashun@imu.edu.cn

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基金项目:

国家重点研发计划(2022YFA0806200)和国家自然科学基金(32030028)资助项目。


The Refinement and Innovation of The UV Cross-linking and Immunoprecipitation
Author:
Affiliation:

1)School of Life Sciences, Inner Mongolia University, Hohhot 010021, China;2)School of Life Sciences, Fudan University, Shanghai 200438, China

Fund Project:

This work was supported by grants from National Key Research and Development Program of China (2022YFA0806200) and The National Natural Science Foundation of China (32030028).

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    摘要:

    RNA结合蛋白(RBPs)在细胞中广泛存在,在多种生物学过程中发挥着重要作用。紫外交联和蛋白质免疫共沉淀(UV cross-linking and immunoprecipitation,CLIP)技术是一种用于鉴定蛋白质直接结合RNA的技术。随着高通量测序技术的发展,CLIP技术已成为鉴定RNA结合蛋白所结合的具体RNA种类和序列的关键方法。为了满足不断增长的科研需求,CLIP技术经历了多次改进,衍生出多种版本,如HITS-CLIP、PAR-CLIP和iCLIP等。日益完善的CLIP技术在揭示新型蛋白质-RNA相互作用、解析RNA修饰和调控机制等方面具有重要价值,在RNA生物学研究中不可或缺。本文详细回顾了这些技术的演进与创新,比较了它们的优势与局限性,探讨了近年来CLIP技术在非经典蛋白质-RNA相互作用的研究中的应用,并展望了CLIP技术在未来RNA生物学中的创新应用与发展方向。

    Abstract:

    RNA-binding proteins (RBPs) are ubiquitous components within cells, fulfilling essential functions in a myriad of biological processes. These proteins interact with RNA molecules to regulate gene expression at various levels, including transcription, splicing, transport, localization, translation, and degradation. Understanding the intricate network of RBP-RNA interactions is crucial for deciphering the complex regulatory mechanisms that govern cellular function and organismal development. Ultravidet (UV) cross-linking and immunoprecipitation (CLIP) stands out as a powerful approach designed to map the precise locations where RBPs bind to RNA. By using UV light to create covalent bonds between proteins and RNA, followed by immunoprecipitation to isolate the protein-RNA complexes, researchers can identify the direct targets of specific RBPs. The advent of high-throughput sequencing technologies has revolutionized CLIP, enabling the identification of not only the types but also the exact sequences of RNA bound by RBPs on a genome-wide scale. The evolution of CLIP has led to the development of specialized variants, each with unique features that address specific challenges and expand the scope of what can be studied. High-throughput sequencing CLIP (HITS-CLIP) was one of the first advancements, significantly increasing the throughput and resolution of RNA-protein interaction mapping. Photoactivatable-ribonucleoside-enhanced CLIP (PAR-CLIP) introduced the use of photoactivatable ribonucleosides to enhance cross-linking efficiency and specificity, reducing background noise and improving the detection of low-abundance RNA-protein interactions. Individual-nucleotide resolution CLIP (iCLIP) further refined the technique, achieving unprecedented precision by resolving individual nucleotides involved in RBP binding, which is particularly valuable for studying the fine details of RNA structure and function. Despite the remarkable progress, there remains room for improvement in CLIP technology. Researchers continue to seek methods to increase sensitivity, reduce technical variability, and improve the reproducibility of results. Advances in sample preparation, data analysis algorithms, and computational tools are critical for addressing these challenges. Moreover, the application of CLIP to more diverse biological systems, including non-model organisms and clinical samples, requires the development of tailored protocols and the optimization of existing ones. Looking forward, the field of RNA biology is poised to benefit greatly from ongoing innovations in CLIP technology. The exploration of non-canonical RNA-protein interactions, such as those involving long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs), promises to reveal new layers of cellular regulation and may lead to the discovery of novel therapeutic targets. Furthermore, integrating CLIP data with other omics approaches, such as proteomics and metabolomics, will provide a more comprehensive understanding of the dynamic interplay between RNA and its binding partners within the cell. In conclusion, the continuous refinement and expansion of CLIP techniques have not only deepened our knowledge of RNA biology but have also opened up new avenues for investigating the molecular underpinnings of health and disease. As the technology matures, it is expected to play an increasingly pivotal role in both basic and applied research, contributing to the advancement of medical science and biotechnology.

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赵佳敏,卢城江,杨明,那顺布和,王纲.紫外交联和蛋白免疫共沉淀技术的演进与创新[J].生物化学与生物物理进展,2025,52(4):1036-1052

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  • 收稿日期:2024-09-11
  • 最后修改日期:2025-03-04
  • 接受日期:2024-11-26
  • 在线发布日期: 2024-12-02
  • 出版日期: 2025-04-28