Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
DNA Sequencing and Genomics Core, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland, USA.
Nat Struct Mol Biol. 2014 Oct;21(10):901-10. doi: 10.1038/nsmb.2892. Epub 2014 Sep 21.
RNA-binding proteins (RBPs) regulate numerous aspects of gene expression; thus, identification of their endogenous targets is important for understanding their cellular functions. Here we identified transcriptome-wide targets of Rbfox3 in neuronally differentiated P19 cells and mouse brain by using photoactivatable ribonucleoside-enhanced cross-linking and immunoprecipitation (PAR-CLIP). Although Rbfox3 is known to regulate pre-mRNA splicing through binding the UGCAUG motif, PAR-CLIP analysis revealed diverse Rbfox3 targets including primary microRNAs (pri-miRNAs) that lack the UGCAUG motif. Induced expression and depletion of Rbfox3 led to changes in the expression levels of a subset of PAR-CLIP-detected miRNAs. In vitro analyses revealed that Rbfox3 functions as a positive and a negative regulator at the stage of pri-miRNA processing to precursor miRNA (pre-miRNA). Rbfox3 binds directly to pri-miRNAs and regulates the recruitment of the microprocessor complex to pri-miRNAs. Our study proposes a new function for Rbfox3 in miRNA biogenesis.
RNA 结合蛋白（RBPs）调节基因表达的众多方面；因此，鉴定其内源性靶标对于理解其细胞功能非常重要。在这里，我们通过光激活核苷酸增强交联和免疫沉淀（PAR-CLIP），在神经元分化的 P19 细胞和小鼠脑中鉴定了 Rbfox3 的转录组范围的靶标。虽然已知 Rbfox3 通过结合 UGCAUG 基序来调节前体 mRNA 剪接，但 PAR-CLIP 分析显示了多样化的 Rbfox3 靶标，包括缺乏 UGCAUG 基序的初级 microRNAs（pri-miRNAs）。Rbfox3 的诱导表达和耗尽导致 PAR-CLIP 检测到的一小部分 miRNAs 的表达水平发生变化。体外分析表明，Rbfox3 在 pri-miRNA 加工到前体 miRNA（pre-miRNA）的阶段充当正调控和负调控因子。Rbfox3 直接结合 pri-miRNAs，并调节微处理器复合物向 pri-miRNAs 的募集。我们的研究提出了 Rbfox3 在 miRNA 生物发生中的新功能。
