Changes in polysome loading as a consequence of RHA downregulation

RNA helicase A (RHA) binds its target transcripts at the post-transcriptional control element (PCE) located in the 5’ untranslated region (UTR). This interaction represents an “RNA switch” that regulates protein synthesis. Down regulation of RHA by siRNAs was used to identify transcripts with RHA-dependent translation. Reduced accumulation of RNA in polysomes was monitored with microarrays. Changes in cytoplasmic RNA steady state abundance was monitored as well. Sixty nine genes exhibit decreased transcript polysome association when subjected to RHA downregulation. A majority of the transcripts that experienced a reduction in the polysome fraction had no significant change in their cytoplasmic abundance (45 genes). Keywords: gene expression array-based Cytoplasmic lysates of cells treated with RHA targeted or non-silencing control siRNAs were separated by sucrose density gradient centrifugation. Ribosomal RNA profiles were generated, fractions containing polysomes were collected, and RNA was extracted.

RNA解旋酶A（RNA helicase A，RHA）可结合位于5'非翻译区（5' untranslated region，UTR）内的转录后调控元件（post-transcriptional control element，PCE），靶向识别其靶转录本。该相互作用构成一种调控蛋白质合成的“RNA开关”。研究人员通过小干扰RNA（siRNA）下调RHA的表达，以鉴定依赖RHA调控翻译的转录本。采用基因芯片（microarray）检测多聚核糖体中RNA的积累量变化，同时同步监测细胞质RNA的稳态丰度改变。结果显示，69个基因的转录本在RHA下调后，其多聚核糖体结合水平显著降低。其中，多聚核糖体组分占比下降的转录本中，有45个的细胞质丰度未发生明显变化。
关键词：基于基因芯片的基因表达分析
实验流程：将经靶向RHA的siRNA或非沉默对照siRNA处理的细胞的细胞质裂解液通过蔗糖密度梯度离心分离；绘制核糖体RNA图谱，收集含有多聚核糖体的组分并提取RNA。