Single base mismatches in the mRNA target site allow specific seed region-mediated off-target binding of siRNA targeting human coagulation factor 7

Abstract

We have analyzed the off-target activity of two siRNAs (F7-1, F7-2) that knock-down human blood coagulation factor 7 mRNA. F7-1 modulates a significant number of non-target transcripts while F7-2 shows high selectivity for the target transcript under various experimental conditions. The 3'-UTRs of all F7-1 off-target genes show statistically significant enrichment of the reverse complement of the F7-1 siRNA seed region located in the guide strand. Seed region enrichment was confirmed in off-target transcripts modulated by siRNA targeting the glucocorticoid receptor. To investigate how these sites contribute to off-target recognition of F7-1, we employed CXCL5 transcript as model system because it contains five F7-1 seed sequence motifs with single base mismatches. We show by transient transfection of reporter gene constructs into HEK293 cells that three out of five sites located in the 3'-UTR region are required for F7-1 off-target activity. For further mechanistic dissection, the sequences of these sites were synthesized and inserted either individually or joined in dimeric or trimeric constructs. Only the fusion constructs were silenced by F7-1 while the individual sites had no off-target activity. Based on F7-1 as a model, a single mismatch between the siRNA seed region and mRNA target sites is tolerated for target recognition and the CXCL5 data suggest a requirement for binding to multiple target sites in off-target transcripts.

Figure 1. Time- and concentration-dependent knock-down of F7 mRNA by F7–1 and F7–2 siRNA in Hep3B cells. (A) Cells were transfected with each siRNA at 10 nM and knock-down efficiency was measured followed 24h, 40h and 48h incubation relative to mRNA levels of lipofectamine transfected cells set to 100%. (B) Cells were transfected with increasing amounts of siRNA and F7 knock-down was measured 24 h after transfection with lipofectamine transfected cells as reference. All quantifications were performed with a commercial branched-DNA assay (Panomics). For details see Materials and Methods.

Figure 2. Time-dependent induction of off-target effects by F7-1 (A) and F7-2 (B) siRNAs in Hep3B cells measured by genome wide microarray profiling. Hep3B cells were transfected with F7-1, F7-2, GFP or luciferase siRNA at 10 nM for the time points indicated above the heatmaps. (A) shows transcripts modulated by F7-1 and the fold change of the same set of transcripts is shown in cells transfected with F7-2, GFP or luciferase siRNA to illustrate specificity for F7-1. (B) shows the equivalent data for F7-2. Expression fold change is displayed as a color gradient from blue (downregulation) to red (upregulation) as indicated by the reference bar. Each line in the heatmap display corresponds to a gene and gene symbols were omitted for clarity. Red asterisk in the heatmap indicate expression levels of F7 for the different conditions while green asterisk indicate expression levels of CXCL5.

Figure 3. siRNA concentration-dependent induction of off-target effects by F7–1 (A) and F7–2 (B) siRNA in Hep3B cells measured by genome wide microarray profiling after 24h treatment. For details see legend of Figure 2.

Figure 4. Detection of F7-1 off-target transcripts in additional human and rat cell lines. The human hepatoma cell lines Hep3B, HepG2 and Huh-7 and the human kidney lines G-401 and HEK293 were transfected with 10 nM F7-1 siRNA for 24 h followed by transcript profiling. In addition, the rat cell lines BRL3A and NRL cells were included to demonstrate species specificity of siRNA off-target effects. Analogous to the experiments shown in Figures 2and3, the transcripts modulated by F7-1 in Hep3B cells were used as reference and the fold change-factor of these genes in the other cell lines is shown in a gradient from blue (downregulation) to red (upregulation) as in Figures 2and3. For the rat cell lines the fold change of the human orthologs present on the rat array is shown. Note that virtually all genes modulated in Hep3B are found in at least one of the other human lines with exception of the upregulated genes.

Figure 5. Enrichment analysis of the F7-1 seed region matches in the 3′-UTR sequence of all expressed genes considering all experimental conditions (A, dose, B, time course, C, human cell lines panel). The ‘Sylamer’ algorithm scans the 3′-UTRs for enrichment of random heptamer motifs. The reverse complement of the F7-1 seed region (red) is significantly enriched in the panel of modulated genes followed by the single mismatch version (5′ CCTGTTC 3′; blue) in most conditions. Enrichment of the 5′ CTATTCA 3′ (black) or 5′ CTGTTCA 3′ (green) heptamers was considered less significant by the program. In the graphic display, the most downregulated genes appear on the left side of the x-axis and induced genes are on the right side of each window. The curve indicates the frequency of each motif and the statistical cut-off is marked by a dotted line. The log10 of the enrichment p value is plotted on the y-axis. Motifs without any significant enrichment in either condition are shown in gray.

Figure 6. Schematic outline of the CXCL5 cloning strategy used for F7–1 binding site mapping. (A) Clone 4 contains the full-length CXCL5 cDNA sequence without poly-A tail. Clones 1–3 represent truncated versions of CXCL5 sequence containing seed motifs A–E with the exact positions indicated in brackets. The coding region of CXCL5 is shown as a gray box. (B) Detection of F7-1 predicted binding sites in CXCL5 cDNA clones by reporter gene assays. The CXCL5 pMIR_REPORT sub-clones 1–4 were transiently co-transfected into HEK293 cells with siRNA and β-galactosidase plasmid for calibration. The data represent the average of three independent experiments with three biological replicates each.