Mastriano Stephen, Kanoria Shaveta, Rennie William, Liu Chaochun, Li Dan, Cheng Jijun, Ding Ye, Lu Jun
Department of Genetics and Yale Stem Cell Center, Yale University, New Haven, CT 06520, USA.
Yale Cancer Center and Center for RNA Science and Medicine, Yale University, New Haven, CT 06520, USA.
bioRxiv. 2024 Dec 6:2024.12.05.626985. doi: 10.1101/2024.12.05.626985.
MicroRNAs (miRNAs) regulate gene expression post-transcriptionally, primarily through binding sites in 3' untranslated regions (3' UTRs). While computational and biochemical approaches have been developed to predict miRNA binding sites on target messenger RNAs, reliable and high-throughput assessment of the regulatory effects of miRNAs on full-length 3' UTRs can still be challenging. Utilizing a miniaturized and high-throughput reporter assay, we present a 'pilot miRNA-targeting map', containing 4,994 successfully measured miRNA:3' UTR regulatory outputs by pairwise assays between 461 miRNAs and eleven 3' UTRs. This collection represents a large experimental miRNA:3' UTR dataset to date on a single platform. The methodology can be generally applied to studies of miRNA-mediated regulation of critical genes. We found that seedless sites can lead to substantial downregulation. We utilized this dataset in the development of a quantitative total score for modeling the total regulatory effects by both seed and seedless sites on a full-length 3' UTR. To assess the predictive value of the total score, we analyzed data from mRNA expression and proteomics studies. We found that the score can discriminate the potent miRNA inhibition from the weak inhibition and is thus useful for quantitative prediction of miRNA regulation. The score has been added to the STarMir program of the Sfold package now available via GitHub at https://github.com/Ding-RNA-Lab/Sfold.
微小RNA（miRNA）主要通过与3'非翻译区（3'UTR）中的结合位点在转录后调节基因表达。虽然已经开发了计算和生化方法来预测miRNA在靶信使RNA上的结合位点，但对miRNA对全长3'UTR的调节作用进行可靠且高通量的评估仍然具有挑战性。利用一种小型化的高通量报告基因检测方法，我们展示了一个“miRNA靶向试验图谱”，其中包含通过461个miRNA与11个3'UTR之间的成对检测成功测量的4994个miRNA:3'UTR调节输出。该集合代表了迄今为止在单个平台上的一个大型实验性miRNA:3'UTR数据集。该方法通常可应用于miRNA介导的关键基因调控研究。我们发现无种子位点可导致显著的下调。我们利用这个数据集开发了一个定量总分，用于模拟种子和无种子位点对全长3'UTR的总调节作用。为了评估总分的预测价值，我们分析了来自mRNA表达和蛋白质组学研究的数据。我们发现该分数可以区分强效miRNA抑制和弱抑制，因此对于miRNA调节的定量预测很有用。该分数现已添加到Sfold软件包的STarMir程序中，可通过GitHub上的https://github.com/Ding-RNA-Lab/Sfold获取。
