2019.01.08,單革教授實驗室發(fā)表文章,報導(dǎo)了其實驗室最新的研究成果。單革實驗室在2017年發(fā)表的Developmental Cell文章基礎(chǔ)上,于Genome Biology發(fā)表了題為《Systematic evaluation of C. elegans lincRNAs with CRISPR knockout mutants》的文章,報導(dǎo)通過優(yōu)化的CRISPR-cas9 系統(tǒng)對秀麗線蟲中155個基因間的長鏈非編碼RNA(lincRNA)進行逐一敲除(秀麗線蟲已知的全部lincRNA共170個),系統(tǒng)地研究了秀麗線蟲中lincRNA的功能。這也是第一篇在多細(xì)胞動物中利用CRISPR-cas9技術(shù)、在全基因組水平上、對一種特定類型長非編碼RNA進行敲除并系統(tǒng)分析其生理功能及功能機理的研究。對155個lincRNA敲除突變體進行六個方面表型的篩選發(fā)現(xiàn)23個lincRNA突變體分別在其中一個或者兩個生理表型上具有不同程度的缺陷。通過對秀麗線蟲不同發(fā)育時期的轉(zhuǎn)錄組測序進行分析,作者研究了lincRNA 和mRNA共表達情況,同時建立了lincRNA和microRNA共表達及調(diào)控網(wǎng)絡(luò)。通過對秀麗線蟲不同發(fā)育時期近300個轉(zhuǎn)錄因子的ChIP-seq分析來探究轉(zhuǎn)錄因子在線蟲不同發(fā)育階段對lincRNA的調(diào)控,進而研究了這23個lincRNA行使其生理調(diào)控功能的機理。本研究系統(tǒng)地探索了lincRNA在多細(xì)胞動物中的生理功能,一定程度拓寬了lincRNA研究領(lǐng)域,同時該研究獲得的lincRNA敲除線蟲株為后續(xù)進一步研究長鏈非編碼RNA在衰老和疾病等中的作用提供了有力支持。文章的共同第一作者是博士生衛(wèi)帥、博士后陳禾、以及國際學(xué)生Emmanuel Enoch Dzakah(最近已博士畢業(yè))。
該研究得到了科技部、國家基金委、以及中科院“衰老的生物學(xué)基礎(chǔ)和干預(yù)策略”先導(dǎo)科技專項(培育)的經(jīng)費支持。
Shuai Wei*, He Chen*, Emmanuel Enoch Dzakah*, Bin Yu, Xiaolin Wang, Tao Fu, Jingxin Li, Lei Liu, Shucheng Fang, Weihong Liu, Ge Shan. Systematic evaluation of C. elegans lincRNAs with CRISPR knockout mutants.Genome Biology, 2019; 20:7.
論文鏈接:https://genomebiology.biomedcentral.com/articles/10.1186/s13059-018-1619-6
Abstract
Long intergenic RNAs (lincRNAs) play critical roles in eukaryotic cells, but systematic analyses of the lincRNAs of an animal for phenotypes are lacking. We generate CRISPR knockout strains for Caenorhabditis elegans lincRNAs and evaluate their phenotypes.
C. elegans lincRNAs demonstrate global features such as shorter length and fewer exons than mRNAs. For the systematic evaluation of C. elegans lincRNAs, we produce CRISPR knockout strains for 155 of the total 170 C. elegans lincRNAs. Mutants of 23 lincRNAs show phenotypes in 6 analyzed traits. We investigate these lincRNAs by phenotype for their gene expression patterns and potential functional mechanisms. Some C. elegans lincRNAs play cis roles to modulate the expression of their neighboring genes, and several lincRNAs play trans roles as ceRNAs against microRNAs. We also examine the regulation of lincRNA expression by transcription factors, and we dissect the pathway by which two transcription factors, UNC-30 and UNC-55, together control the expression of linc-73. Furthermore, linc-73 possesses a cis function to modulate the expression of its neighboring kinesin gene unc-104 and thus plays roles in C. elegans locomotion.
By using CRISPR/cas9 technology, we generate knockout strains of 155 C. elegans lincRNAs as valuable resources for studies in noncoding RNAs, and we provide biological insights for 23 lincRNAs with the phenotypes identified in this study.
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