Wright Jane E, Gaidatzis Dimos, Senften Mathias, Farley Brian M, Westhof Eric, Ryder Sean P, Ciosk Rafal
Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland.
EMBO J. 2011 Feb 2;30(3):533-45. doi: 10.1038/emboj.2010.334. Epub 2010 Dec 17.
RNA-binding proteins (RBPs) are critical regulators of gene expression. To understand and predict the outcome of RBP-mediated regulation a comprehensive analysis of their interaction with RNA is necessary. The signal transduction and activation of RNA (STAR) family of RBPs includes developmental regulators and tumour suppressors such as Caenorhabditis elegans GLD-1, which is a key regulator of germ cell development. To obtain a comprehensive picture of GLD-1 interactions with the transcriptome, we identified GLD-1-associated mRNAs by RNA immunoprecipitation followed by microarray detection. Based on the computational analysis of these mRNAs we generated a predictive model, where GLD-1 association with mRNA is determined by the strength and number of 7-mer GLD-1-binding motifs (GBMs) within UTRs. We verified this quantitative model both in vitro, by competition GLD-1/GBM-binding experiments to determine relative affinity, and in vivo, by 'transplantation' experiments, where 'weak' and 'strong' GBMs imposed translational repression of increasing strength on a non-target mRNA. This study demonstrates that transcriptome-wide identification of RBP mRNA targets combined with quantitative computational analysis can generate highly predictive models of post-transcriptional regulatory networks.
RNA结合蛋白（RBPs）是基因表达的关键调节因子。为了理解和预测RBP介导的调控结果，有必要对它们与RNA的相互作用进行全面分析。RBPs的RNA信号转导与激活（STAR）家族包括发育调节因子和肿瘤抑制因子，如秀丽隐杆线虫的GLD-1，它是生殖细胞发育的关键调节因子。为了全面了解GLD-1与转录组的相互作用，我们通过RNA免疫沉淀结合微阵列检测来鉴定与GLD-1相关的mRNA。基于对这些mRNA的计算分析，我们生成了一个预测模型，其中GLD-1与mRNA的结合由UTR内7聚体GLD-1结合基序（GBMs）的强度和数量决定。我们通过竞争GLD-1/GBM结合实验在体外验证了这个定量模型，以确定相对亲和力，在体内通过“移植”实验进行验证，其中“弱”和“强”GBMs对非靶标mRNA施加了强度递增的翻译抑制。这项研究表明，全转录组范围内对RBP mRNA靶标的鉴定结合定量计算分析能够生成转录后调控网络的高度预测模型。
