The LncRNA NEAT1 Nests RNA Binding Proteins and the Microprocessor to Globally Enhance Pri-miRNA Processing. Homo sapiens

MicroRNA biogenesis is known to be modulated by a variety of RNA binding proteins (RBPs), but in most cases, individual RBPs appear to influence the processing of a small number of selective targets. We herein report binding of the NONO/PSF heterodimer to hundreds of expressed pri-miRNAs in HeLa cells to globally enhance pri-miRNA processing by the Drosha/DGCR8 Microprocessor. As NONO/PSF are key components of paraspeckles organized by the lncRNA NEAT1, we find that NEAT1 also has profound effects on global pri-miRNA processing. Mechanistic dissection reveals that NEAT1 broadly interacts with NONO/PSF as well as many other RBPs, and that multiple RNA segments in NEAT1, including a “pseudo pri-miRNA” near its 3’ end, help attract the Microprocessor. These findings suggest a bird nest model for a large lncRNA to orchestrate efficient processing of an entire class of small RNAs in the nucleus.we used small RNA-seq to identify miRNA level in response to secific knockdowns relative to siGFP treatment control Overall design: We profiled the miRNAs expression levels under conditions including siGFP(control), siNEAT1, siNEAT1_V2, siNONO, siPSF and siPSPC1 through small RNA-seq respectively. And we also examined the in vivo interactions between NONO/PSF and RNA through CLIP-seq.

已知多种RNA结合蛋白（RNA binding proteins, RBPs）可调控微小RNA（MicroRNA, miRNA）的生物发生过程，但在绝大多数情况下，单个RNA结合蛋白仅会影响少量选择性靶标的加工。本研究报道了NONO/PSF异二聚体在海拉（HeLa）细胞中与数百种表达的初级miRNA（pri-miRNA）结合，通过Drosha/DGCR8微处理器（Microprocessor）全局性增强初级miRNA的加工效率。鉴于NONO/PSF是由长链非编码RNA（long non-coding RNA, lncRNA）NEAT1组装形成的旁斑小体（paraspeckles）的关键组分，我们发现NEAT1对全局初级miRNA加工也具有显著调控作用。机制解析结果显示，NEAT1可广泛结合NONO/PSF以及多种其他RNA结合蛋白，且NEAT1内部的多个RNA区段——包括其3'端附近的"伪初级miRNA"——有助于招募微处理器复合物。上述研究发现提出了一种“鸟巢模型”，即大型长链非编码RNA可在细胞核内协调一整类小RNA的高效加工。我们通过小RNA测序（small RNA-seq）鉴定了相对于siGFP处理对照组的特异性敲低样本中的miRNA表达水平。实验整体设计：我们分别通过小RNA测序分析了siGFP（对照组）、siNEAT1、siNEAT1_V2、siNONO、siPSF及siPSPC1处理条件下的miRNA表达水平；同时我们还通过交联免疫沉淀测序（CLIP-seq）检测了NONO/PSF与RNA在体内的相互作用。