Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis.
Mapping Argonaute and conventional RNA-binding protein interactions with RNA at single-nucleotide resolution using HITS-CLIP and CIMS analysis.
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DOI：
10.1038/nprot.2014.012
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发表时间：
2014-02
期刊：
影响因子：
14.8
通讯作者：
中科院分区：
文献类型：
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作者：
确定RNA结合蛋白（RNABP）与靶RNA相互作用的位点为理解RNA调控的巨大复杂性打开了大门。紫外线交联和免疫沉淀（CLIP）是一种变革性技术，其中从体内交联的RNA-蛋白质复合物中纯化的RNA被测序以揭示RNABP：RNA接触的足迹。CLIP结合高通量测序（HITS-CLIP）是一种可推广的策略，可以产生比标准RNA免疫沉淀（RIP）分析或纯计算方法更高准确度和分辨率的全转录组RNA结合图谱。将CLIP应用于Argonaute蛋白质已经扩展了这种方法的实用性，以绘制microRNA和其他小调控RNA的结合位点。最后，数据分析的最新进展利用交联诱导突变位点（CIMS）将RNA结合图谱细化到单核苷酸分辨率。一旦IP条件建立，HITS-CLIP需要大约八天的时间来制备用于测序的RNA。建立数据分析管道，包括CIMS，需要3-4天。
Identifying sites where RNA binding proteins (RNABPs) interact with target RNAs opens the door to understanding the vast complexity of RNA regulation. UV-crosslinking and immunoprecipitation (CLIP) is a transformative technology in which RNAs purified from in vivo cross-linked RNA-protein complexes are sequenced to reveal footprints of RNABP:RNA contacts. CLIP combined with high throughput sequencing (HITS-CLIP) is a generalizable strategy to produce transcriptome-wide RNA binding maps with higher accuracy and resolution than standard RNA immunoprecipitation (RIP) profiling or purely computational approaches. Applying CLIP to Argonaute proteins has expanded the utility of this approach to mapping binding sites for microRNAs and other small regulatory RNAs. Finally, recent advances in data analysis take advantage of crosslinked-induced mutation sites (CIMS) to refine RNA-binding maps to single-nucleotide resolution. Once IP conditions are established, HITS-CLIP takes approximately eight days to prepare RNA for sequencing. Established pipelines for data analysis, including for CIMS, take 3-4 days.
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影响因子：
64.8
作者：
Baek, Daehyun;Villen, Judit;Shin, Chanseok;Camargo, Fernando D.;Gygi, Steven P.;Bartel, David P.
通讯作者：
Bartel, David P.
影响因子：
64.5
作者：
Hafner M;Landthaler M;Burger L;Khorshid M;Hausser J;Berninger P;Rothballer A;Ascano M Jr;Jungkamp AC;Munschauer M;Ulrich A;Wardle GS;Dewell S;Zavolan M;Tuschl T
通讯作者：
Tuschl T
影响因子：
16.8
作者：
通讯作者：
--
影响因子：
14.9
作者：
Gäken J;Mohamedali AM;Jiang J;Malik F;Stangl D;Smith AE;Chronis C;Kulasekararaj AG;Thomas NS;Farzaneh F;Tavassoli M;Mufti GJ
通讯作者：
Mufti GJ
影响因子：
16.2
作者：
Ince-Dunn G;Okano HJ;Jensen KB;Park WY;Zhong R;Ule J;Mele A;Fak JJ;Yang C;Zhang C;Yoo J;Herre M;Okano H;Noebels JL;Darnell RB
通讯作者：
Darnell RB