Comprehensive and quantitative mapping of RNA-protein interactions across a transcribed eukaryotic genome
Comprehensive and quantitative mapping of RNA-protein interactions across a transcribed eukaryotic genome
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DOI：
10.1073/pnas.1618370114
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发表时间：
2017-04-04
影响因子：
11.1
通讯作者：
Greenleaf, William J.
中科院分区：
文献类型：
--
作者：
She, Richard;Chakravarty, Anupam K.;Greenleaf, William J.
RNA结合蛋白（RBP）控制着细胞中几乎所有转录物的命运。然而，没有现有的方法来研究这些转录后基因调控相结合的转录组范围的吞吐量和生物物理精度。在这里，我们描述了一种实现这一点的测定。使用常用的硬件，我们构建了一个可定制的开源平台，利用Illumina技术的固有吞吐量进行直接生物物理测量。我们使用该平台来定量测量原型RBP Vts1对酿酒酵母基因组中每个转录物的结合亲和力。这些测量的规模和精度揭示了许多以前未知的功能，这种良好的研究RBP。我们的转录基因组阵列（TGA）以同等的熟练程度检测了稀有和丰富的转录本，揭示了数百个被以前的方法错过的低丰度靶点。这些靶点调节多种生物学过程，包括营养感应和DNA损伤反应，并涉及Vts1在从头基因的诞生。TGA为每个结合位点提供了单核苷酸分辨率，并描绘了Vts1结合的高度特异性序列和结构基序。vts1中转录水平的变化。细胞建立了这些结合位点的调节功能。Vts1对转录本丰度的影响在很大程度上与其在mRNA中的结合位置无关，这对RBP如何驱动RNA降解的流行假设提出了挑战。因此，TGA能够在转录组范围内定量描述变体RNA结构、亲和力和体内表型之间的关系。我们预计，TGA将提供类似的全面和定量的见解，几乎任何RBP的功能。
RNA-binding proteins (RBPs) control the fate of nearly every transcript in a cell. However, no existing approach for studying these posttranscriptional gene regulators combines transcriptome-wide throughput and biophysical precision. Here, we describe an assay that accomplishes this. Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the Saccharomyces cerevisiae genome. The scale and precision of these measurements revealed many previously unknown features of this well-studied RBP. Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in de novo gene "birth." TGA provided single-nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding. Changes in transcript levels in vts1. cells established the regulatory function of these binding sites. The impact of Vts1 on transcript abundance was largely independent of where it bound within an mRNA, challenging prevailing assumptions about how this RBP drives RNA degradation. TGA thus enables a quantitative description of the relationship between variant RNA structures, affinity, and in vivo phenotype on a transcriptome-wide scale. We anticipate that TGA will provide similarly comprehensive and quantitative insights into the function of virtually any RBP.