Systematic evaluation of C. elegans lincRNAs with CRISPR knockout mutants [lncRNA-seq]. Systematic evaluation of C. elegans lincRNAs with CRISPR knockout mutants [lncRNA-seq]

Background: LincRNAs play critical roles in eukaryotic cells, but systematic analyses of lincRNAs of an animal for phenotypes have been missing. We have generated CRISPR knockout strains for C. elegans lincRNAs and have evaluated their phenotypes Results: C. elegans lincRNAs demonstrate global features such as shorter length and fewer exons than mRNAs. For the systematic evaluation of C. elegans lincRNAs, CRISPR knockout strains for 155 out of all the 170 C. elegans lincRNAs were produced. Mutants of 23 lincRNAs have shown phenotypes in 6 traits analyzed. We have investigated these phenotypic lincRNAs for their gene expression patterns and their potential functional mechanisms. Some C. elegans lincRNAs play cis roles to modulate the expression of their neighboring genes, and some other lincRNAs play trans roles as ceRNAs against microRNAs. LincRNAs are extensively regulated by transcription factors, and we dissect the pathway that two transcription factors UNC-30 and UNC-55 control the expression of linc-73 together. Furthermore, linc-73 plays a cis role to modulate the expression of its neighboring gene unc-104, and thus regulates the formation of presynapses. Conclusions: By using CRISPR/cas9 technology, we have generated knockout strains of 155 C. elegans lincRNAs as valuable resources for studies in noncoding RNAs, and we provide biological insights for 23 phenotypic lincRNAs identified from 6 traits examined. Overall design: C. elegans mRNA profiles of 9 different stages (N2_embryo, N2_L1, N2_L2, N2_L3, N2_L4, N2_Young_Adult, N2_dauer, N2_starved_mixture, him-5) were generated by deep sequencing, using Illumina HiSeq 2500 and BGISEQ-500.

研究背景：基因间长链非编码RNA（long intergenic non-coding RNA，lincRNA）在真核细胞中发挥关键调控作用，但目前尚未有针对动物源lincRNA开展系统性表型分析的研究报道。本研究针对秀丽隐杆线虫（Caenorhabditis elegans，C. elegans）的lincRNA构建了CRISPR（成簇规律间隔短回文重复序列）敲除菌株，并对其表型进行了系统评估。
研究结果：相较于信使RNA（messenger RNA，mRNA），秀丽隐杆线虫的lincRNA具有长度更短、外显子数目更少的全局特征。为系统性评估该线虫的lincRNA功能，我们成功构建了170个已知秀丽隐杆线虫lincRNA中的155个的CRISPR敲除菌株。在本次分析的6项表型指标中，23个lincRNA的敲除突变体呈现出显著表型差异。我们进一步针对这些具有表型的lincRNA开展了基因表达模式分析与潜在作用机制研究。部分秀丽隐杆线虫lincRNA通过顺式作用调控邻近基因的表达，另有部分lincRNA作为内源竞争RNA（competing endogenous RNA，ceRNA）通过反式作用靶向拮抗微小RNA（microRNA，miRNA）。lincRNA的表达广泛受转录因子调控，本研究还解析了转录因子UNC-30与UNC-55共同调控linc-73表达的信号通路。此外，linc-73可通过顺式作用调控邻近基因unc-104的表达，进而参与突触前体的形成调控。
研究结论：本研究借助CRISPR/Cas9技术构建了155个秀丽隐杆线虫lincRNA的敲除菌株，可为非编码RNA领域的相关研究提供宝贵的实验资源；同时针对6项表型筛选中获得的23个具有表型的lincRNA，本研究提供了重要的生物学见解。
整体实验设计：我们采用Illumina HiSeq 2500与BGISEQ-500测序平台开展深度测序，获取了秀丽隐杆线虫9个不同发育阶段的mRNA表达谱，具体样本包括N2胚胎、N2 L1幼虫、N2 L2幼虫、N2 L3幼虫、N2 L4幼虫、N2年轻成虫、N2 dauer滞育态、N2饥饿混合样本以及him-5突变体。