Investigating the role of Dicer-dependent transfer RNA-derived small RNAs in gene expression regulation. Investigating the role of Dicer-dependent transfer RNA-derived small RNAs in gene expression regulation

We used sequencing analyses to identify small RNAs that are generated in a Dicer-dependent manner and the genes that are dysregulated upon Dicer knockdown at the mRNA level, and upon Drosha, Dicer and Ago2 knockdown at the chromatin level in HEK293 cells. We found that levels of tRNA-derived sRNAs decreased alongside microRNAs upon Dicer knockdown. Many genes are dysregulated at the chromatin levels when Dicer and Ago2 levels are reduced, indicating that Dicer and Ago2 can regulate genes directly or indirectly at the transcriptional level. Drosha knockdown, on the other hand, has a marginal effect on RNA levels at the chromatin. Overall design: sRNA and mRNA profiles of HEK293 wt and Dicer knockdown cells were generated by deep sequencing, in triplicates; chromatin-associated RNA profiles of HEK293 wt, Drosha, Dicer and Ago2 knockdown cells were generated by deep sequencing, in duplicates.

本研究通过测序分析，在HEK293细胞中鉴定出以Dicer核糖核酸酶(Dicer)依赖性方式生成的小RNA(small RNAs, sRNAs)，同时鉴定出两类失调基因：一类是在mRNA水平经Dicer核糖核酸酶敲低后出现失调的基因，另一类是在染色质水平经Drosha核糖核酸酶(Drosha)、Dicer核糖核酸酶及Ago2(Ago2蛋白)敲低后出现失调的基因。研究发现，在Dicer核糖核酸酶敲低后，tRNA来源的小RNA(tRNA-derived sRNAs)与微RNA(microRNAs, miRNAs)的表达水平均出现下调。当Dicer核糖核酸酶与Ago2蛋白的表达水平降低时，诸多基因在染色质水平上出现失调，这表明Dicer核糖核酸酶及Ago2蛋白可在转录层面直接或间接调控基因表达。与之相对，Drosha核糖核酸酶敲低对染色质层面的RNA水平仅存在微弱影响。
实验整体设计：通过深度测序(deep sequencing)对HEK293野生型(HEK293 wt)与Dicer核糖核酸酶敲低细胞的小RNA及mRNA表达谱进行检测，设置三次生物学重复；同时通过深度测序对HEK293野生型、Drosha核糖核酸酶敲低、Dicer核糖核酸酶敲低及Ago2蛋白敲低细胞的染色质结合RNA表达谱进行检测，设置两次生物学重复。