CLIP-related methodologies and their application to retrovirology.
CLIP-related methodologies and their application to retrovirology.
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DOI：
10.1186/s12977-018-0417-2
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发表时间：
2018-05-02
期刊：
影响因子：
3.3
通讯作者：
Kutluay SB
中科院分区：
文献类型：
--
作者：
Bieniasz PD;Kutluay SB
事实上，HIV-1复制的每一步和许多细胞抗病毒防御机制都受到病毒或细胞RNA结合蛋白（RBP）与HIV-1 RNA上不同序列或结构元件结合的调节。直到最近，这些蛋白质-RNA相互作用的研究主要是在体外结合试验与遗传学方法的补充。然而，这些方法在生理相关环境中识别RBP的相关靶标方面受到高度限制。交联免疫沉淀测序（CLIP）方法的发展彻底改变了蛋白质-核酸复合物的分析。CLIP将共价交联蛋白质-RNA复合物的免疫沉淀与高通量测序相结合，以接近核苷酸的分辨率提供了细胞（或病毒体）中感兴趣的RBP结合的RNA序列的全球账户。CLIP协议的许多变体最近被开发出来，其中一些在原始版本上有重大改进。在此，我们简要回顾这些方法，并给出CLIP已成功应用于逆转录病毒学研究的例子。
Virtually every step of HIV-1 replication and numerous cellular antiviral defense mechanisms are regulated by the binding of a viral or cellular RNA-binding protein (RBP) to distinct sequence or structural elements on HIV-1 RNAs. Until recently, these protein–RNA interactions were studied largely by in vitro binding assays complemented with genetics approaches. However, these methods are highly limited in the identification of the relevant targets of RBPs in physiologically relevant settings. Development of crosslinking-immunoprecipitation sequencing (CLIP) methodology has revolutionized the analysis of protein–nucleic acid complexes. CLIP combines immunoprecipitation of covalently crosslinked protein–RNA complexes with high-throughput sequencing, providing a global account of RNA sequences bound by a RBP of interest in cells (or virions) at near-nucleotide resolution. Numerous variants of the CLIP protocol have recently been developed, some with major improvements over the original. Herein, we briefly review these methodologies and give examples of how CLIP has been successfully applied to retrovirology research.
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