Flamand Mathieu N, Gan Hin Hark, Mayya Vinay K, Gunsalus Kristin C, Duchaine Thomas F
Department of Biochemistry & Goodman Cancer Research Centre, McGill University, Montreal, Quebec H3G 1Y6 Canada.
Center for Genomics and Systems Biology, Department of Biology, New York University, 12 Waverly Place, New York, NY 10003, USA.
Nucleic Acids Res. 2017 Jul 7;45(12):7212-7225. doi: 10.1093/nar/gkx340.
Although strong evidence supports the importance of their cooperative interactions, microRNA (miRNA)-binding sites are still largely investigated as functionally independent regulatory units. Here, a survey of alternative 3΄UTR isoforms implicates a non-canonical seedless site in cooperative miRNA-mediated silencing. While required for target mRNA deadenylation and silencing, this site is not sufficient on its own to physically recruit miRISC. Instead, it relies on facilitating interactions with a nearby canonical seed-pairing site to recruit the Argonaute complexes. We further show that cooperation between miRNA target sites is necessary for silencing in vivo in the C. elegans embryo, and for the recruitment of the Ccr4-Not effector complex. Using a structural model of cooperating miRISCs, we identified allosteric determinants of cooperative miRNA-mediated silencing that are required for both embryonic and larval miRNA functions. Our results delineate multiple cooperative mechanisms in miRNA-mediated silencing and further support the consideration of target site cooperation as a fundamental characteristic of miRNA function.
尽管有力的证据支持了它们协同相互作用的重要性，但微小RNA（miRNA）结合位点在很大程度上仍被作为功能独立的调控单元进行研究。在此，对可变3΄UTR异构体的一项调查表明，在协同miRNA介导的沉默中存在一个非经典的无种子位点。虽然该位点对于靶mRNA的去腺苷酸化和沉默是必需的，但它自身并不足以物理性地招募miRISC。相反，它依赖于促进与附近经典种子配对位点的相互作用来招募AGO蛋白复合体。我们进一步表明，miRNA靶位点之间的协同作用对于秀丽隐杆线虫胚胎中的体内沉默以及Ccr4-Not效应复合体的招募是必需的。利用协同miRISC的结构模型，我们确定了协同miRNA介导的沉默的变构决定因素，这些因素对于胚胎期和幼虫期的miRNA功能都是必需的。我们的结果描绘了miRNA介导的沉默中的多种协同机制，并进一步支持将靶位点协同作用视为miRNA功能的一个基本特征。
