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

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-argeted 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. mRNA expression profiling by RNA-seq of cerebellum and cortex from wild-type (WT), Cyrano miR-7 site mutant (CyrMut), Cyrano-/- (CyrKO), and Mir7a1-/-; Mir7b-/- (Mir7DKO) mice. This study consists of 33 polyA-selected stranded NEXTflex libraries prepared from 3-4 biological replicates for each tissue and each genotype. To minimize batch effects, libraries for wild-type tissues were prepared and sequenced for each experiment and only intra-experiment comparisons were made.非编码RNA（Noncoding RNAs, ncRNAs）的基因调控功能日益受到学界重视。本研究描述了以四种非编码RNA为核心的调控网络——包含一种长链非编码RNA（long ncRNA）、一种环状RNA（circular RNA）以及两种微小RNA（microRNAs, miRNAs）——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所产生的分子效应。长链非编码RNA Cyrano通过一段广泛互补配对的位点结合miR-7，进而触发该微小RNA的降解。Cyrano介导的miR-7降解效率远高于此前已报道的靶向引导微小RNA降解案例，此类案例均基于人工RNA与病毒RNA的相关研究。通过降低miR-7的表达水平，Cyrano可解除其对miR-7靶向mRNA的抑制作用，从而促进环状RNA Cdr1as的积累——该环状RNA已被证实可调控神经元活动。若缺乏Cyrano，过量的miR-7会引发Cdr1as在细胞质内的降解，这一过程部分源于第二种微小RNA miR-671对Cdr1as的剪切作用增强。综上，多种类型的非编码RNA可协同构建一套精密复杂的调控网络。本研究对野生型（WT）、Cyrano miR-7位点突变型（CyrMut）、Cyrano基因敲除型（CyrKO）以及Mir7a1-/-; Mir7b-/-（Mir7DKO）小鼠的小脑与大脑皮层组织开展了基于RNA-seq的mRNA表达谱分析。本研究共构建33个经polyA富集的链特异性NEXTflex文库，每个组织与基因型均设置3至4个生物学重复样本。为最大限度降低批次效应，野生型组织的文库均在同一次实验中完成构建与测序，本研究仅开展实验内的样本比较分析。