Distinct MUNC lncRNA structural domains regulate transcription of different promyogenic factors [SHAPE-MaP]

Many lncRNAs have been discovered using transcriptomic data, however, it is unclear what fraction of lncRNAs is functional and what structural properties affect their phenotype. MUNC lncRNA (also known as DRReRNA) acts as an enhancer RNA for the Myod1 gene in cis and stimulates the expression of other promyogenic genes in trans by recruiting the cohesin complex. Here, experimental probing of the RNA structure revealed that MUNC contains multiple structural domains not detected by prediction algorithms in the absence of experimental information. We show that these specific and structurally distinct domains are required for induction of promyogenic genes, for binding genomic sites and gene expression regulation, and for binding the cohesin complex. Myod1 induction and cohesin interaction comprise only a subset of MUNC phenotype. Our study reveals unexpectedly complex, structure-driven functions for the MUNC lncRNA and emphasizes the importance of experimentally determined structures for understanding structure-function relationships in lncRNAs. Overall design: SHAPE-MaP on RNA from C2C12 cells overexpressing MUNC variants for a total 16 samples (8 DMSO, 85NIA): Cell-free SHAPE of 2 MUNC spliced replicates, MUNC genomic, CH1 deletion, and CH4 deletion; In-cell SHAPE of 2 replicates of MUNC spliced and 1 replicate of MUNC genomic

目前已通过转录组数据发现诸多长链非编码RNA（lncRNA，long non-coding RNA），但尚不明确其中具备功能的比例，以及哪些结构特性会影响其表型。
MUNC lncRNA（亦称DRReRNA）可在顺式作用中作为Myod1基因的增强子RNA，并通过招募黏连蛋白复合物（cohesin complex）在反式作用中激活其他促肌基因的表达。本研究通过对RNA结构进行实验探测发现，MUNC拥有多个结构域，而在无实验数据支撑的情况下，预测算法无法识别这些结构域。我们证实，这些具有特异性且结构各异的结构域是激活促肌基因、结合基因组位点并调控基因表达，以及结合黏连蛋白复合物所必需的。Myod1基因激活与黏连蛋白结合仅为MUNC表型的一部分。本研究揭示了MUNC lncRNA此前未被认知的、由结构驱动的复杂功能，并强调了实验解析的结构对于理解长链非编码RNA中结构-功能关系的重要性。
整体实验设计：对过表达MUNC变体的C2C12细胞的RNA进行SHAPE-MaP（Selective 2'-羟基酰化修饰引物延伸测序，SHAPE-MaP）实验，共设置16个样本（8个二甲基亚砜（dimethyl sulfoxide, DMSO）对照组、85NIA处理组）：分别对2份MUNC剪接体重复样本、MUNC基因组转录本、CH1缺失突变体及CH4缺失突变体进行无细胞体系SHAPE实验；对2份MUNC剪接体重复样本及1份MUNC基因组转录本样本进行细胞内SHAPE实验。