Genomewide view of gene silencing by small interfering RNAs

Abstract

RNA interference (RNAi) is an evolutionarily conserved mechanism in plant and animal cells that directs the degradation of messenger RNAs homologous to short double-stranded RNAs termed small interfering RNA (siRNA). The ability of siRNA to direct gene silencing in mammalian cells has raised the possibility that siRNA might be used to investigate gene function in a high throughput fashion or to modulate gene expression in human diseases. The specificity of siRNA-mediated silencing, a critical consideration in these applications, has not been addressed on a genomewide scale. Here we show that siRNA-induced gene silencing of transient or stably expressed mRNA is highly gene-specific and does not produce secondary effects detectable by genomewide expression profiling. A test for transitive RNAi, extension of the RNAi effect to sequences 5' of the target region that has been observed in Caenorhabditis elegans, was unable to detect this phenomenon in human cells.

Fig. 1.

Silencing of a model gene by siRNAs. (A) Silencing of transiently expressed and integrated GFP gene by siRNAs. Sequences of the siRNAs used are indicated on the left. For silencing of transiently transfected GFP, 0.3 μgof pGFP was transfected with 1 μg of pSEAP2-control and 12 picomoles of the indicated siRNA in HEK293 cells. For silencing of an integrated GFP gene, HEK293-derived Phoenix cells expressing GFP after retroviral transduction (Materials and Methods) were transfected with the 12 picomoles of the indicated siRNA and 1 μg of pSEAP2-control. GFP expression was determined by FACS 48 h (transient GFP target) or 72 h (integrated GFP target) after transfection. The mean fluorescence intensity was normalized for transfection efficiency by the alkaline phosphatase activity of pSEAP2-control (Materials and Methods). The experiments were done in triplicate, and the means (± standard deviation) of GFP fluorescence intensity relative to mock transfected cells (no siRNA) are shown. (B) Fluorescence photomicroscopy and FACS plots of cells stably expressing GFP and transfected with the indicated siRNAs.

Fig. 2.

Global gene expression changes associated with RNAi. (A) Summary of gene expression data. Global gene expression patterns in three siRNA experiments were analyzed; in each set, the gene expression of cells that were mock transfected (no siRNA), transfected with GFP siRNA, or cognate control siRNA were determined in parallel in triplicate. Data sets: E1, HEK293 cells with transiently expressed GFP target treated with E1, C1, or no siRNA; E2, HEK293 cells with transiently expressed GFP target treated with E2, C2, or no siRNA; stable, Phoenix cells stably expressing an integrated GFP gene treated with E1, C1, or no siRNA. Genes that had signal intensity >1.5-fold of the local spot element background in the reference channel and were present for >80% of the data set were considered well measured. The number of well measured genes are shown on the second column; these genes were analyzed in the multiclass comparison by using SAM (15). The number of genes that had an estimated FDR of <0.05 and the FDR of the top 10 performing genes for each data set are shown on the right two columns. (B) Minimal gene expression changes associated with siRNA-mediated RNAi. The 10 genes with the most consistent changes in expression in response to the experimental manipulation, in each of the three siRNA experiments, were collated into a nonredundant gene list. The expression changes of this group of genes in all experiments are displayed in matrix format (16). The expression ratios were mean-centered within each data set, and the gene expression changes are indicated by the color scale as indicated below.

Fig. 3.

Test of transitive RNAi in HEK293 cells. (A) Experimental strategy for transitive RNAi. The square indicates the original trigger siRNA, and the dashed lines indicate secondary siRNAs. The regions of GFP targeted by E1 and E3 are indicated by arrows. (B) Effect of siRNAs (E1 and E3) on expression of GFP fusion constructs. HEK293 cells were transfected with the indicated constructs and siRNAs and photographed by fluorescence microscopy 48 h after transfection. (C) Effect of siRNAs on luciferase-actin expression. The luciferase activity in cells transfected with the indicated constructs and siRNA (E1 or E3) were compared with those of corresponding constructs and control siRNA (C1); the values shown are the means of relative activity (E1/C1 or E3/C1) ± standard deviation of triplicate experiments.