MicroRNA调控Notch信号转导的分子机制

MicroRNAs (miRNAs) are small RNAs that negatively regulate target gene expression in many developmental and metabolic processes. Increasing evidence suggest that critical genes targeted by miRNAs in multiple signaling pathways regulate cell fate specification, tissue patterning and organ formation. One of such signaling processes is the Notch pathway, which is at the core of cell fate decisions in development and adult tissue homeostatis, ranging from Drosophila wing morphogenesis to vertebrate cardiovascular development and neurogenesis. To interrogate the role of miRNA on Notch signaling and to uncover novel Notch signaling effectors that are subject to regulation by miRNA, we performed a gain-of-function screen to identify novel miRNAs that modulate Notch signaling in Drosophila. We identified a highly conserved miRNA, Drosophila miRNA 932 (dmir-932), that potentially regulates Notch signaling. Most importantly, knocking down six novel genes that are predicted to be the potential dmir-932 targets resulted in stereotypical Notch signaling defects. These results led us to hypothesize that dmir-932 plays an essential role in Notch signaling. Here, we propose to dissect the molecular mechanism by which dmir-932 regulates Notch signaling, with a special emphasis on the function of two of the six candidate dmir-932 targets: Rab3-GEF and CG42613. This objective will be achieved by the following specific aims: 1) Determine the role of dmir-932 in Notch signaling and identify bona fide targets of dmir-932; 2) Investigate how Rab3-GEF, a regulator of Rab GTPases, controls membrane trafficking processes of key components of the Notch signaling cascade; 3) Examine the regulatory role of CG42613, which encodes a low density lipoprotein receptor (LDLR) domain-containing protein, in Notch signaling. Given that Notch signaling is highly conserved from insects to humans, and as Notch signaling plays important roles in development and many key cellular processes in postnatal tissues, the proposed studies of the normal physiological roles of novel miRNAs and their targets in Notch signaling, in a relatively simple genetic model system, are anticipated to be helpful for developing novel approaches to prevent and treat birth defects and cancer.

MicroRNA(miRNA)在转录后水平对信号转导途径中的核心元件进行负向修饰，从而调控细胞命运决定，组织形成和器官发生。保守的Notch信号广泛地参与从果蝇到哺乳动物发育的一系列过程。为探索miRNA及其修饰的靶基因在Notch途径中的作用，本实验室在果蝇中进行了miRNA的过表达筛选，发现高度保守的dmir-932及其6个预测靶基因参与Notch信号的调节。因此提出项目申请，计划从以下三方面展开研究：1）确定dmir-932及其靶基因对Notch信号转导途径的调控；2）研究靶基因Rab3-GEF通过膜运输来调控Notch途径；3）探索靶基因低密度脂蛋白受体CG42613对Notch信号的作用。由于Notch在发育和成体细胞中的高度保守性，揭示miRNA及其靶基因Rab3-GEF和CG42613在Notch途径的调控机制，将为与Notch相关疾病和癌症更为有效的诊断和治疗提供理论基础

MicroRNA（miRNA）在转录后水平对信号转导途径中的核心元件进行负向修饰，从而调控细胞命运决定，组织形成和器官发生。我们在果蝇翅成虫盘中开展了miRNA过表达筛选，观察其对Notch，Hedgehog（Hh）和Wingless（Wg）信号通路的影响，发现多个可调节发育信号转导的miRNA，并对其中三个miRNA以及靶基因ubpy、stuxnet（stx）、mago nashi（mago）进行深入的研究。第一项研究发现ubpy是miR-279的靶基因。去泛素化酶UBPY调控包括Hh信号通路核心蛋白Smoothened在内的多个需要经过泛素化修饰的膜蛋白的细胞内定位。UBPY通过调控转运必需内体分选复合体（Endosomal sorting complex required for transport，ESCRT）的稳态，从而综合调控多个发育信号通路的全新机制。这项工作已在Development上发表（Zhang et al., 2014）。第二项研究表明stx是miR-932的靶基因。stx与编码多梳蛋白抑制复合体1（PRC1）核心成分Polycomb（Pc）存在遗传互作。Stx蛋白特异性结合并调控Pc蛋白在蛋白酶体的降解，从而在表观遗传水平解除其对Notch受体基因的抑制。进一步研究表明Stx对Pc蛋白的调控机理在哺乳动物中高度保守。这项工作以封面文章的形式发表在Developmental Cell（Du et al., 2016）。第三项研究表明mago是miR-35的靶基因。mago编码外显子接合复合体 (Exon Junction Complex, EJC) 的一个核心蛋白。遗传学实验显示EJC参与调节Wg信号通路。EJC通过调节基底层细胞极性决定复合物的关键因子Discs large 1（Dlg1）的mRNA剪接和转录水平，特异性地调节支架蛋白Disheveled（Dsh）的稳定性，进而确保信号接收细胞能接收和感知存在于细胞外间质的Wg信号。相关工作已在eLIFE上发表（Liu et al., 2016）。终上所述，我们对miRNA及其靶基因的研究揭示了表观遗传、RNA剪接和蛋白质降解在发育中起重要作用的Notch、Hh和Wg等信号通路上的关键作用。由于这些调节机制在演化上高度保守，我们在果蝇上的研究为相关疾病和癌症更为有效的诊断和治疗提供理论基础。