PDF(Pubmed)
Abstract：
Although Argonaute (AGO) proteins have been the focus of microRNA (miRNA) studies, we observed AGO-free mature miRNAs directly interacting with RNA-binding proteins, implying the sophisticated nature of fine-tuning gene regulation by miRNAs. To investigate microRNA-binding proteins (miRBPs) globally, we analyzed PAR-CLIP data sets to identify RBP quaking (QKI) as a novel miRBP for let-7b. Potential existence of AGO-free miRNAs were further verified by measuring miRNA levels in genetically engineered AGO-depleted human and mouse cells. We have shown that QKI regulates miRNA-mediated gene silencing at multiple steps, and collectively serves as an auxiliary factor empowering AGO2/let-7b-mediated gene silencing. Depletion of QKI decreases interaction of AGO2 with let-7b and target mRNA, consequently controlling target mRNA decay. This finding indicates that QKI is a complementary factor in miRNA-mediated mRNA decay. QKI, however, also suppresses the dissociation of let-7b from AGO2, and slows the assembly of AGO2/miRNA/target mRNA complexes at the single-molecule level. We also revealed that QKI overexpression suppresses cMYC expression at post-transcriptional level, and decreases proliferation and migration of HeLa cells, demonstrating that QKI is a tumour suppressor gene by in part augmenting let-7b activity. Our data show that QKI is a new type of RBP implicated in the versatile regulation of miRNA-mediated gene silencing.
摘要：
尽管Argonaute（AGO）蛋白一直是microRNA（miRNA）研究的焦点，我们观察到无AGO的成熟miRNA与RNA结合蛋白直接相互作用，暗示着miRNA微调基因调控的复杂性质。为了在全球范围内研究microRNA结合蛋白（miRBPs），我们分析了PAR-CLIP数据集，以确定RBP震颤（QKI）是let-7b的新型miRBP。通过测量遗传工程化的AGO耗尽的人和小鼠细胞中的miRNA水平进一步验证了无AGOmiRNA的潜在存在。我们已经表明，QKI在多个步骤中调节miRNA介导的基因沉默，并共同充当增强AGO2/let-7b介导的基因沉默的辅助因素。QKI的消耗降低了AGO2与let-7b和靶mRNA的相互作用，从而控制目标mRNA衰减。这一发现表明QKI是miRNA介导的mRNA衰变的互补因子。QKI,然而,还抑制了let-7b与AGO2的解离，并在单分子水平上减缓了AGO2/miRNA/靶mRNA复合物的组装。我们还发现QKI过表达在转录后水平抑制cMYC表达，减少HeLa细胞的增殖和迁移，证明QKI是一种抑癌基因，部分增强了let-7b的活性。我们的数据表明，QKI是一种新型的RBP，参与miRNA介导的基因沉默的通用调控。
