A network of noncoding regulatory RNAs acts in the mammalian brain III

Noncoding RNAs (ncRNAs) play increasingly appreciated gene-regulatory roles. Here, we describe a regulatory network centered on four ncRNAs--a long ncRNA, a circular RNA, and two microRNAs--using gene editing in mice to probe the molecular consequences of disrupting key components of this network. The long ncRNA Cyrano uses an extensively paired site to miR-7 to trigger destruction of this microRNA. Cyrano-directed miR-7 degradation is much more efficient than previously described examples of target-directed microRNA degradation, which come from studies of artificial and viral RNAs. By reducing miR-7 levels, Cyrano prevents repression of miR-7-targeted mRNAs and enables the accumulation of Cdr1as, a circular RNA known to regulate neuronal activity. Without Cyrano, excess miR-7 causes cytoplasmic destruction of Cdr1as, in part through enhanced slicing of Cdr1as by a second miRNA, miR-671. Thus, several types of ncRNAs can collaborate to establish a sophisticated regulatory network. miRNA expression profiling by small-RNA sequencing of cerebellum and hippocampus from wild-type (WT) and Cyrano-/- (CyrKO) mice, and of cerebellum from Cdr1as miR-671 site mutant (Cdr1as671), and double mutant (CyrKO;Cdr1as671) mice. This study consists of 18 libraries generated from 3 biological replicates per genotype for each tissue.非编码RNA（Noncoding RNAs, ncRNAs）的基因调控作用日益受到学界重视。本研究聚焦由四种非编码RNA构成的调控网络——包含一条长链非编码RNA、一条环状RNA以及两种微小RNA（microRNA, miRNA）——通过在小鼠体内开展基因编辑实验，探究该网络关键组分被破坏后产生的分子效应。长链非编码RNA Cyrano通过与miR-7形成广泛配对的结合位点，介导该微小RNA的降解。Cyrano介导的miR-7降解效率远高于此前基于人工RNA与病毒RNA研究报道的靶标导向型微小RNA降解案例。通过降低miR-7的表达水平，Cyrano可解除miR-7靶向mRNA的翻译抑制，进而促进已知可调控神经元活动的环状RNA Cdr1as的积累。若缺乏Cyrano，过量的miR-7会引发Cdr1as在细胞质中的降解，其中部分机制是第二种微小RNA miR-671对Cdr1as的剪切作用增强。综上，多种类型的非编码RNA可协同作用，构建出精密复杂的调控网络。本研究通过小RNA测序技术，对野生型（wild-type, WT）、Cyrano基因敲除（Cyrano-/-，简称CyrKO）小鼠的小脑与海马体，以及Cdr1as的miR-671结合位点突变型（Cdr1as671）、双突变型（CyrKO;Cdr1as671）小鼠的小脑组织进行miRNA表达谱分析。本研究共构建18个测序文库，每个组织对应每种基因型设置3次生物学重复。