Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.
Comprehensive analysis of RNA-protein interactions by high-throughput sequencing-RNA affinity profiling.
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DOI：
10.1038/nmeth.2970
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发表时间：
2014-06
期刊：
影响因子：
48
通讯作者：
Lis, John T.
中科院分区：
文献类型：
--
作者：
Tome, Jacob M.;Ozer, Abdullah;Pagano, John M.;Gheba, Dan;Schroth, Gary P.;Lis, John T.
RNA-蛋白质相互作用在基因调控中具有关键作用。然而，缺乏高通量的方法来定量分析这些相互作用。我们采用Illumina GAIIx测序仪，用高重复测序- RNA亲和力分析（HiTS-RAP）测定进行数百万次这样的测量。数以百万计的cDNA被测序，并被E. coli复制终止子蛋白Tus，并原位转录，于是Tus停止转录，留下稳定附着于其模板DNA的RNA。然后在测序仪中定量荧光标记蛋白的结合。我们使用HiTS-RAP来测量GFP结合适体和NELF-E结合适体的诱变文库对其各自靶标的亲和力，从而鉴定了两种适体中对其RNA-蛋白质相互作用至关重要的区域。我们发现，突变的叠加影响NELF-E结合适体的结合亲和力，其相互作用主要取决于单链RNA基序，但不影响GFP适体的结合亲和力，其相互作用主要取决于二级结构。
RNA-protein interactions have critical roles in gene regulation. However, high-throughput methods to quantitatively analyze these interactions are lacking. We adapted an Illumina GAIIx sequencer to make several million such measurements with a High-Throughput Sequencing – RNA Affinity Profiling (HiTS-RAP) assay. Millions of cDNAs are sequenced, bound by the E. coli replication terminator protein Tus, and transcribed in situ, whereupon Tus halts transcription leaving RNA stably attached to its template DNA. The binding of fluorescently-labeled protein is then quantified in the sequencer. We used HiTS-RAP to measure the affinity of mutagenized libraries of GFP-binding and NELF-E binding aptamers to their respective targets and thereby identified regions in both aptamers that are critical for their RNA-protein interaction. We show that mutations additively affect binding affinity of the NELF-E binding aptamer, whose interaction depends mainly on a single-stranded RNA motif, but not that of the GFP aptamer, whose interaction depends primarily on secondary structure.
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影响因子：
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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
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