Signature transcript 3′ UTRs contain sequence complementarity to siRNA seed regions. The indicated siRNAs were transfected into HeLa cells. Gene expression signatures (p-value ≤ 0.01) for each siRNA were determined 24 h post-transfection. Down-regulated transcripts are indicated in light blue, up-regulated transcripts are indicated in magenta. Gray indicates no significant change in transcript level in response to the siRNA. The sense sequence for each siRNA is indicated, with nucleotides complementary to positions 2–7 of the seed region of the guide strand indicated in dark blue. Positions 1 and 8 of the guide strand are indicated in light blue to highlight the entire seed region of the siRNA. The lines below the sequences indicate the positions of the three seed region hexamers. The 3′ UTRs of signature transcripts were searched for complementarity to siRNA seed region hexamers. Shown are the e-values (p-value with Bonferroni correction) for down-regulated transcripts with 3′ UTR complementarity to each seed region hexamer. Also shown is the percentage of down-regulated, up-regulated, or background transcripts containing 3′ UTR complementarity to the hexamer.

shRNA and siRNA of the same sequence regulate the same subset of seed-region-complementary transcripts. HT29 cells were transduced with lentivirus expressing PLK1 shRNA under control of a doxycycline-inducible promoter. PLK1 transcript silencing at times following induction of the shRNA was measured by TaqMan qPCR (upper left panel). The gene expression signature resulting from 24-h induction of PLK1 shRNA is shown (upper right panel). Transcripts down-regulated by the shRNA are indicated in light blue; transcripts up-regulated by the shRNA are indicated in magenta. Down-regulated transcripts with 3′ UTRs that contain sequence complementarity to the shRNA seed region are indicated as dark blue dots. A PLK1 siRNA of the same sequence as the shRNA was transfected into HeLa cells, and gene expression signatures were generated at 24 h post-transfection (lower left panel). The transcripts down-regulated by the PLK1 shRNA were highlighted in the siRNA signature and are indicated as dark blue dots. A PLK1 siRNA of a different sequence was transfected into HeLa cells (lower right panel). The transcripts regulated by the PLK1 shRNA are indicated as dark blue dots.

siRNA seed regions preferentially align with the 3′ UTR regions of unintended transcripts. (Upper panels) 36 siRNAs were aligned by FASTA to their down-regulated off-target signature transcripts and to a background set of 10,631 transcript sequences with mapped CDS. The longest contiguous stretch of identity and the transcript region targeted in each guide-strand alignment were determined. Shown on the x-axis is the length of contiguous sequence alignment between the siRNAs and the transcripts they silence. Shown on the y-axis is the frequency within the signature set (left panel) or background set (right panel) of alignments of the length indicated on the x-axis. (Bottom panels) The positions of sequence complementarity between the siRNA guide strand and the target transcript were determined by FASTA alignment. Shown on the x-axis is the position in the siRNA guide strand, with 1 indicating the first position at the 5′ end of the guide strand. Shown on the y-axis is the number of perfect matches at each position of the siRNA to transcripts in the signature set (left panel) or background set (right panel).

Position-specific effect of base mismatches on silencing of off-target transcripts. (A) siRNAs to MAPK14–193 were synthesized to contain a single base substitution at each position. siRNAs were then transfected into HeLa cells, and changes in transcript regulation were analyzed by microarray profiling (Jackson et al. 2003). Shown is a heatmap representing 52 genes regulated (x-axis) in 61 experiments (y-axis). These 52 transcripts represent the consensus signature of down-regulated transcripts for the MAPK14 siRNA. Transcripts shown were regulated with p ≤ 0.01. No cuts were placed on fold regulation. Transcripts down-regulated in siRNA-transfected cells are shown in light blue, transcripts up-regulated in siRNA-transfected cells are shown in magenta. Black indicates no change in transcript level. Three independent experiments are shown for each base mismatch. The gold box indicates the location of the siRNA seed region (positions 2–8 of the guide strand). Transcripts are ordered by percent change in down-regulation (normalized mean log ratio change) across the wild-type signature. The arrow indicates the location of the target transcript MAPK14. (B) siRNAs to MAPK14–193 containing mismatches at position 1 or position 2 of the seed region were transfected into HeLa cells, and changes in transcript regulation were analyzed by microarray as described in A.

Off-target protein regulation by siRNAs. Decreasing concentrations of an siRNA targeting PIK3CB were transfected into HeLa cells. Silencing of the intended target (PIK3CB) or two off-target transcripts previously identified by microarray profiling (FADD, YY1) was measured by TaqMan analysis at 24 h post-transfection. In parallel samples, protein silencing was measured by Western blotting at 48 h post-transfection. (Top panel) Sequence identity of the siRNA with PIK3CB (on-target), FADD and YY1 (off-target) transcripts. Shown are the sense strand sequence for the siRNA and the transcript sequence for the silenced transcripts. Red font denotes positions of identity. (Middle panel) Western blotting data from a representative experiment. (Bottom panel) Quantitative analysis of mRNA silencing (solid lines) and protein silencing (dashed lines) versus siRNA dose. mRNA levels were normalized to HGUS levels and protein levels were normalized to actin levels. Data are expressed as a percentage of levels in mock-transfected cells. The scatter graphs show means ±SD of 3–4 independent experiments.