Freeberg Mallory A, Han Ting, Moresco James J, Kong Andy, Yang Yu-Cheng, Lu Zhi John, Yates John R, Kim John K
Genome Biol. 2013 Feb 14;14(2):R13. doi: 10.1186/gb-2013-14-2-r13.
Protein-RNA interactions are integral components of nearly every aspect of biology, including regulation of gene expression, assembly of cellular architectures, and pathogenesis of human diseases. However, studies in the past few decades have only uncovered a small fraction of the vast landscape of the protein-RNA interactome in any organism, and even less is known about the dynamics of protein-RNA interactions under changing developmental and environmental conditions.
Here, we describe the gPAR-CLIP (global photoactivatable-ribonucleoside-enhanced crosslinking and immunopurification) approach for capturing regions of the untranslated, polyadenylated transcriptome bound by RNA-binding proteins (RBPs) in budding yeast. We report over 13,000 RBP crosslinking sites in untranslated regions (UTRs) covering 72% of protein-coding transcripts encoded in the genome, confirming 3' UTRs as major sites for RBP interaction. Comparative genomic analyses reveal that RBP crosslinking sites are highly conserved, and RNA folding predictions indicate that secondary structural elements are constrained by protein binding and may serve as generalizable modes of RNA recognition. Finally, 38% of 3' UTR crosslinking sites show changes in RBP occupancy upon glucose or nitrogen deprivation, with major impacts on metabolic pathways as well as mitochondrial and ribosomal gene expression.
Our study offers an unprecedented view of the pervasiveness and dynamics of protein-RNA interactions in vivo.
蛋白质 - RNA相互作用几乎是生物学各个方面不可或缺的组成部分，包括基因表达调控、细胞结构组装以及人类疾病的发病机制。然而，在过去几十年里，在任何生物体中，对蛋白质 - RNA相互作用组这一广阔领域的研究仅揭示了一小部分，对于在不断变化的发育和环境条件下蛋白质 - RNA相互作用的动态变化了解更少。
在此，我们描述了gPAR - CLIP（全局光活化核糖核苷增强交联和免疫纯化）方法，用于捕获芽殖酵母中与RNA结合蛋白（RBP）结合的非翻译、多聚腺苷酸化转录组区域。我们报告了超过13000个位于非翻译区（UTR）的RBP交联位点，覆盖了基因组中72%的蛋白质编码转录本，证实3' UTR是RBP相互作用的主要位点。比较基因组分析表明，RBP交联位点高度保守，RNA折叠预测表明二级结构元件受蛋白质结合的限制，可能作为RNA识别的通用模式。最后，38%的3' UTR交联位点在葡萄糖或氮缺乏时显示出RBP占据情况的变化，对代谢途径以及线粒体和核糖体基因表达有重大影响。
我们的研究提供了体内蛋白质 - RNA相互作用的普遍性和动态性的前所未有的观点。
