2019.01.08，單革教授實(shí)驗(yàn)室發(fā)表文章，報(bào)導(dǎo)了其實(shí)驗(yàn)室最新的研究成果。單革實(shí)驗(yàn)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(bào)導(dǎo)通過優(yōu)化的CRISPR-cas9 系統(tǒng)對(duì)秀麗線蟲中155個(gè)基因間的長(zhǎng)鏈非編碼RNA(lincRNA)進(jìn)行逐一敲除（秀麗線蟲已知的全部lincRNA共170個(gè)），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(dòng)物中利用CRISPR-cas9技術(shù)、在全基因組水平上、對(duì)一種特定類型長(zhǎng)非編碼RNA進(jìn)行敲除并系統(tǒng)分析其生理功能及功能機(jī)理的研究。對(duì)155個(gè)lincRNA敲除突變體進(jìn)行六個(gè)方面表型的篩選發(fā)現(xiàn)23個(gè)lincRNA突變體分別在其中一個(gè)或者兩個(gè)生理表型上具有不同程度的缺陷。通過對(duì)秀麗線蟲不同發(fā)育時(shí)期的轉(zhuǎn)錄組測(cè)序進(jìn)行分析，作者研究了lincRNA 和mRNA共表達(dá)情況，同時(shí)建立了lincRNA和microRNA共表達(dá)及調(diào)控網(wǎng)絡(luò)。通過對(duì)秀麗線蟲不同發(fā)育時(shí)期近300個(gè)轉(zhuǎn)錄因子的ChIP-seq分析來(lái)探究轉(zhuǎn)錄因子在線蟲不同發(fā)育階段對(duì)lincRNA的調(diào)控，進(jìn)而研究了這23個(gè)lincRNA行使其生理調(diào)控功能的機(jī)理。本研究系統(tǒng)地探索了lincRNA在多細(xì)胞動(dòng)物中的生理功能，一定程度拓寬了lincRNA研究領(lǐng)域，同時(shí)該研究獲得的lincRNA敲除線蟲株為后續(xù)進(jìn)一步研究長(zhǎng)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(guó)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(guó)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(xiàng)（培育）的經(jīng)費(fèi)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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