Drosha knockdown in human cells reduces viability but does not affect snRNA or rRNA synthesis or processing. Homo sapiens

We asked whether the human drosha protein, an RNase III homolog known to process microRNAs (miRNAs), might also be a small nuclear RNA (snRNA) 3' processing factor. Using retroviral siRNA silencing constructs, we stably knocked down drosha protein to nearly undetectable levels. Knockdown cells exhibited reduced growth rates and viability compared to controls, but no accumulation of unprocessed U2 snRNA precursors. In fungi, RNase III homologs process rRNA precursors and certain mRNAs. Although rRNA processing appears to be normal in the drosha knockdown cells, expression microarray analysis revealed misregulation of several mRNAs involved in cell growth and proliferation. Curiously, drosha knockdown appeared to downregulate the predicted mRNA targets of several miRNAs Keywords: siRNA knockdown Overall design: The experimental goal was to evaluate gene expression changes induced by siRNA knockdown of drosha. Four samples of HeLa cells were transfected with retroviral siRNA expression vectors: two replicates of an anti-GFP siRNA vector (siGFP) and two different anti-drosha siRNA vectors (sidroshaB and sidroshaC). Cells were selected with puromycin 24 hours after transfection and harvested 72 hours after transfection. Trizol-harvested RNA was processed with standard Affymetrix protocols and hybridized to U133Plus2.0 GeneChips. Signals were scaled to an arbitrary global mean value of 800.

我们旨在探究，已知可加工微RNA（miRNAs）的核糖核酸酶III（RNase III）同源蛋白——人类Drosha蛋白，是否同时可作为小核RNA（snRNA）的3'端加工因子。我们采用逆转录病毒介导的小干扰RNA（siRNA）沉默构建体，将Drosha蛋白稳定敲低至近乎无法检测的水平。与对照组相比，敲低细胞的生长速率与细胞活力均出现下降，但未检测到未加工U2 snRNA前体的积累。在真菌中，核糖核酸酶III同源蛋白负责加工核糖体RNA（rRNA）前体与特定信使RNA（mRNA）。尽管Drosha敲低细胞内的rRNA加工过程看似正常，但表达微阵列分析结果显示，多个参与细胞生长与增殖过程的mRNA出现表达失调。值得注意的是，Drosha敲低似乎下调了多种miRNA的预测靶标mRNA。关键词：小干扰RNA（siRNA）敲低 整体实验设计：本实验旨在评估siRNA敲低Drosha所诱导的基因表达变化。将4组HeLa细胞转染逆转录病毒siRNA表达载体：其中两组为抗绿色荧光蛋白（GFP）siRNA载体（siGFP）的生物学重复，另外两组为两种不同的抗Drosha siRNA载体（sidroshaB与sidroshaC）。转染24小时后使用嘌呤霉素进行细胞筛选，并于转染72小时后收获细胞。采用Trizol法提取的RNA按照标准Affymetrix实验流程进行处理，并与U133Plus2.0基因芯片进行杂交。信号值经标准化至任意全局均值800。