Figure 4. CFIm25/68 is a natural PCPA activator.

(A) Western blot analysis of HeLa cell lysates transfected with control, CFIm25, or CFIm68 siRNA. The knockdown efficiencies relative to Actin as a loading control are indicated as percentages of residual protein for each knockdown compared to control. (B) Genome browser views of 4-thiouridine-labeled RNA-seq data with indicated siRNA knockdowns in HeLa cells on representative genes (GLS, ACACA, and SIAH1). The Y-axis indicates RPM for the highest peak within the genome browser field for each RNA-seq. Annotated RefSeq gene structures are shown in blue with thin horizontal lines indicating introns and thicker blocks indicating exons. The 5’ss is indicated by a black arrow above the gene structure. Green arrows indicate major peaks showing natural PCPA within 1kb downstream of the first 5’ss. We note that the increase in the PCPA peak is readily apparent by comparison to the nearby peak in exon1. (C) Box plots showing the distribution of RNA-seq read counts in the 1kb downstream of the intron to the reads the first exon upon siRNA knockdown or U1 AMO. The median of the data is indicated as a notch in the box, while the whisker depicts 1.5 times the inter- quartile range, and outliers are shown as dots. The significance of difference between each knockdown group was performed using Wilcoxon rank sum test, all the P-values are <2.2 × 10⁻¹⁶.