(a) Distribution of shRNA potency in functionally distinct transcript regions. Shown is the potency distribution of shRNAs in the unbiased TILE data set that target the 5’UTR, CDS or 3’UTR. Since these shRNAs were evaluated using the Sensor assay, their targets are not subject to alternative cleavage and polyadenylation (ApA) and/or splicing events.
(b) AU content of potent and weak miR-30 shRNAs from the unbiased TILE set. Potent shRNAs tend to have a higher proportion of A/U nucleotides (p < 2.2e-16, two-sided Kolmogorov-Smirnov test).
(c) AU content of functionally distinct transcript regions in the human genome. Shown are the AU densities in 5’UTR, CDS and 3’UTR.
(d) AU content in mouse transcripts.
(e) Alternative cleavage and polyadenylation (ApA) prevents potent shRNAs from inhibiting their putative target gene. Immunoblotting of Pten in NIH/3T3s transduced at single-copy with LEPG expressing the indicated shRNAs. Nine top predictions targeting the CDS or the 3’UTR after early ApA sites were compared alongside controls for their ability to suppress mouse Pten. Actb was used as loading control.
(f) Comparison of knockdown efficiency and annotation of ApA sites. Shown are potent Pten shRNA predictions and their position (start, end) on the mouse genome (mm9). KD indicates a qualitative degree of the knockdown observed in immunoblotting analyses of NIH/3T3s (e). ApA indicates previously published positions on the mouse genome (mm9) of ApA sites (alternative 3’ ends) identified in NIH/3T3 and mouse ES cells by 3P-Seq. 2P-Seq shows the quantification of transcript expression levels measured by 2P-Seq. All shRNAs and ApA sites are ordered according to their position along the mouse genome.