Fig 2: CFIm controls Pten mRNA 3′UTR isoform expression. (A) Representative Pten tracks of 3′UTR-seq upon CFIm25, -59 and -68 KD in NIH3T3. Schematic of Pten and 3′UTR isoforms previously captured by 3′ RACE (13) is shown at the bottom. Each track is separated into two parts with different scales indicated as count ranges on top to show all major isoforms. (B) Quantitative northern blotting of endogenous Pten upon CFIm KD in NIH3T3. Corresponding 3′UTR isoforms are indicated on the right with the size markers on the left. Bar graph on the bottom tabulates the average relative composition of the 300 nt, 3.3k and 5-6k isoforms across three biological replicates. (C) Pten 3′UTR isoform-specific qRT-PCR for the 300 nt, 3.3k and 5-6k isoforms upon CFIm KD in NIH3T3. (D) CFIm59 and CFIm68 expression rescue in respective KO cells. (E) Pten 3′UTR isoform ratio shift upon CFIm59 and CFIm68 rescue. APA 300 nt and APA 3.3k levels are measured by qRT-PCR and taken as ratios before normalization to the mock transfection control. (F) 3′UTR-seq tracks of Pten upon CFIm59 and CFIm68 rescue. All experiments were performed in at least three biological replicates with technical triplicates for qRT-PCR. Error bars indicated are mean ± standard deviation. P-values were calculated with one-way ANOVA followed by Dunnett's test in (C) and with Student's t-test in (E) (*P≤ 0.05, **P≤ 0.01, ***P≤ 0.001, ****P≤ 0.0001, and ns not significant).

Fig 3: CFIm APA regulation in the PI3K/Akt pathway. (A) Tabulated APA status of genes designated as part of the PI3K/Akt pathway by KEGG, or the Akt signalling cascade by GO, from our NIH3T3 3′UTR-seq. No APA: genes with no detectable alterative 3′UTR isoform. CFIm NR: genes not responsive to CFIm KD. CFIm Resp: genes with detectable switch in 3′UTR isoform usage upon CFIm KD. (B) Schematic of the PI3K/Akt cascades. Genes responsive to CFIm KD are circled and shaded in gray, with example genes in brackets. Genes non-responsive to CFIm KD but with more than one detectable 3′UTR isoform are circled without shading. Genes with no detectable APA are not circled nor shaded. (C, D) RED scores (C) and 3'UTR-seq gene tracks (D) of core PI3K/Akt components Pten, Pik3ca, Pik3r3 and Akt3, upon CFIm KD in NIH3T3.

Fig 4: CFIm regulates Pten mRNA and protein expression. (A) Representative western blots of CFIm25, -59 and -68 KD and the corresponding change in PTEN level. Two different siRNAs were used for each KD, and the efficiency was quantified. Actin was used as a loading control. (B) Normalized quantification of PTEN protein expression upon CFIm KD. (C) Quantification of normalized total Pten mRNA expression upon CFIm KD assayed through qRT-PCR and normalized to Hprt1. (D) Western blotting of CFIm25, -59 and -68 showing co-dependent protein expression. All error bars indicate mean ± standard deviation across at least three biological replicates. P-values were calculated with one-way ANOVA followed by Dunnett's test (*P≤ 0.05, **P≤ 0.01, ***P≤ 0.001 and ****P≤ 0.0001).

Fig 5: Distinct and opposing roles for CFIm59 and CFIm68 on global APA. (A) Up- and downregulated distal and proximal PAS counts upon CFIm KD or KO. Analyses of our NIH3T3 3′ mRNA-seq and public datasets of HEK293T PAS-seq were performed to tabulate each PAS expression change upon CFIm disruption. Statistical significance was calculated with DEXSeq or Fisher's exact test and corrected for multiple testing. Dark gray indicates PAS differential expression with false discovery rate (FDR) ≤0.05 and light gray for FDR >0.05. (B) Relative expression difference (RED) score distribution of NIH3T3 and HEK293T upon CFIm disruption. RED of each gene was calculated as the log2 difference between KD (or KO) distal/proximal and control distal/proximal ratio. Paired two-tailed Student's t-test was used (****P≤ 0.0001). (C) Overlap of 3′UTRs shortened upon CFIm68 KD and lengthened upon CFIm59 KD in NIH3T3. (D, E) Genome tracks of example cancer-related mRNAs whose 3'UTRs lengthen upon CFIm59 depletion and shorten upon CFIm25 and -68 depletion in NIH3T3 (D) and HEK293T (E) cells.