Journal: Nucleic Acids Research

Article Title: Distinct, opposing functions for CFIm59 and CFIm68 in mRNA alternative polyadenylation of Pten and in the PI3K/Akt signalling cascade

doi: 10.1093/nar/gkac704

Figure Lengend Snippet: PTEN APA in cancers. ( A ) PDUI analyses of PTEN and AKT3 in prostate cancer and glioblastoma. Each cancer is divided into transcriptomic subtypes defined by The Cancer Genome Atlas (TCGA) project as shown in the bottom legend. PDUIs of cancer datasets were collated from The Cancer 3′UTR Atlas (TC3A). PDUIs of non-cancer prostate and brain tissues were mined from APAatlas. ( B, C ) Spearman correlations of CFIm59 and CFIm68 mRNA level with PTEN expression ( B ), and of PTEN , CFIm59 and CFIm68 mRNAs with PTEN PDUI ( C ) across 34 cancer types (detailed in Materials and Methods).

Article Snippet: Knockdown in NIH3T3 was performed by reverse transfection of siRNAs using Lipofectamine RNAiMAX (ThermoFisher) with a final concentration of 10 nM for 72 h. The following siRNAs were used: negative ctrl (Qiagen 1027281), CFIm25-si1 (Qiagen SI04414732), CFIm25-si2 (Qiagen SI04414739), CFIm59-si1 (Qiagen SI00858655), CFIm59-si2 (forward: 5′-GUCCUCAUCUCCUCUCUUATT-3′, reverse: 5′-UAAGAGAGGAGAUGAGGACTT-3′), CFIm68-si1 (Qiagen SI00958741) and CFIm68-si2 (Qiagen SI00958748).

Techniques: Expressing

Journal: Nucleic Acids Research

Article Title: Distinct, opposing functions for CFIm59 and CFIm68 in mRNA alternative polyadenylation of Pten and in the PI3K/Akt signalling cascade

doi: 10.1093/nar/gkac704

Figure Lengend Snippet: 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 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).

Article Snippet: Knockdown in NIH3T3 was performed by reverse transfection of siRNAs using Lipofectamine RNAiMAX (ThermoFisher) with a final concentration of 10 nM for 72 h. The following siRNAs were used: negative ctrl (Qiagen 1027281), CFIm25-si1 (Qiagen SI04414732), CFIm25-si2 (Qiagen SI04414739), CFIm59-si1 (Qiagen SI00858655), CFIm59-si2 (forward: 5′-GUCCUCAUCUCCUCUCUUATT-3′, reverse: 5′-UAAGAGAGGAGAUGAGGACTT-3′), CFIm68-si1 (Qiagen SI00958741) and CFIm68-si2 (Qiagen SI00958748).

Techniques: Expressing, Northern Blot, Quantitative RT-PCR, Transfection, Control, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Distinct, opposing functions for CFIm59 and CFIm68 in mRNA alternative polyadenylation of Pten and in the PI3K/Akt signalling cascade

doi: 10.1093/nar/gkac704

Figure Lengend Snippet: CFIm subunits and UGUA RNA elements in Pten APA regulation. ( A ) Schematic of five UGUA motifs surrounding mouse Pten PAS 300 nt and PAS 3.3k on a CMV-driven construct. Distance between the PAS and each UGUA is indicated. Bottom shows individual constructs with mutations introduced at each UGUA site, and the regions swapped in the experiments featured in (F). ( B, D ) Mouse Pten -specific northern blotting of HEK293T wildtype (B), CFIm59 KO and CFIm68 KO cells (D) transfected with Pten UGUA mutant constructs. Size markers are indicated on the left and the Pten 3′UTR isoforms on the right. Empty: mock transfection with construct backbone only. ( C, E ) Quantification of (B) and (D) across three biological replicates. Ratio of 300 nt and 3.3k isoforms was calculated for each transfected sample and normalized to the ratio of wildtype (WT) Pten -transfected sample. ( F ) Mouse Pten- specific northern blotting (left) and qRT-PCR (right) on Pten wildtype and swap constructs transfected in HEK293T. qRT-PCR quantification was averaged over biological triplicates. Error bars are mean ± standard deviation and P -values were calculated with one-way ANOVA followed by Dunnett's test (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001, and ns not significant).

Article Snippet: Knockdown in NIH3T3 was performed by reverse transfection of siRNAs using Lipofectamine RNAiMAX (ThermoFisher) with a final concentration of 10 nM for 72 h. The following siRNAs were used: negative ctrl (Qiagen 1027281), CFIm25-si1 (Qiagen SI04414732), CFIm25-si2 (Qiagen SI04414739), CFIm59-si1 (Qiagen SI00858655), CFIm59-si2 (forward: 5′-GUCCUCAUCUCCUCUCUUATT-3′, reverse: 5′-UAAGAGAGGAGAUGAGGACTT-3′), CFIm68-si1 (Qiagen SI00958741) and CFIm68-si2 (Qiagen SI00958748).

Techniques: Construct, Northern Blot, Transfection, Mutagenesis, Quantitative RT-PCR, Standard Deviation

Journal: Nucleic Acids Research

Article Title: Distinct, opposing functions for CFIm59 and CFIm68 in mRNA alternative polyadenylation of Pten and in the PI3K/Akt signalling cascade

doi: 10.1093/nar/gkac704

Figure Lengend Snippet: Model of Pten APA regulation by CFIm. ( A ) CFIm59 and -68 depletion both upregulate PTEN protein, but through different effects on APA. Because long isoforms (APA 3.3k and APA 5-6k) are better translated than the short, a ∼3-fold upregulation of long isoforms upon CFIm59 KD leads to a similar (∼1.5-fold) PTEN protein increase as a ∼10-fold increase in the short (APA 300) isoform upon CFIm68 KD. ( B, C ) CFIm25/CFIm59 complex promotes usage of APA 300 nt more efficiently (solid arrow) than APA 3.3k (dashed arrow), whereas CFIm25/CFIm68 is a better promoter of APA 3.3k than APA 300 nt. In the presence of both CFIm59 and -68 ( B ), loss of UGUA motif around either major PAS leads to APA shift in favor of the other PAS. In the absence of either CFIm59 or -68 ( C ) the same trend of APA shift occurs with UGUA mutations due to the partial compensation between the two complexes, but the magnitude depends on the targeting specificity of the complex present.

Article Snippet: Knockdown in NIH3T3 was performed by reverse transfection of siRNAs using Lipofectamine RNAiMAX (ThermoFisher) with a final concentration of 10 nM for 72 h. The following siRNAs were used: negative ctrl (Qiagen 1027281), CFIm25-si1 (Qiagen SI04414732), CFIm25-si2 (Qiagen SI04414739), CFIm59-si1 (Qiagen SI00858655), CFIm59-si2 (forward: 5′-GUCCUCAUCUCCUCUCUUATT-3′, reverse: 5′-UAAGAGAGGAGAUGAGGACTT-3′), CFIm68-si1 (Qiagen SI00958741) and CFIm68-si2 (Qiagen SI00958748).

Techniques:

Journal: Nucleic Acids Research

Article Title: Distinct, opposing functions for CFIm59 and CFIm68 in mRNA alternative polyadenylation of Pten and in the PI3K/Akt signalling cascade

doi: 10.1093/nar/gkac704

Figure Lengend Snippet: 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 log 2 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.

Article Snippet: Knockdown in NIH3T3 was performed by reverse transfection of siRNAs using Lipofectamine RNAiMAX (ThermoFisher) with a final concentration of 10 nM for 72 h. The following siRNAs were used: negative ctrl (Qiagen 1027281), CFIm25-si1 (Qiagen SI04414732), CFIm25-si2 (Qiagen SI04414739), CFIm59-si1 (Qiagen SI00858655), CFIm59-si2 (forward: 5′-GUCCUCAUCUCCUCUCUUATT-3′, reverse: 5′-UAAGAGAGGAGAUGAGGACTT-3′), CFIm68-si1 (Qiagen SI00958741) and CFIm68-si2 (Qiagen SI00958748).

Techniques: Expressing, Disruption, Control, Two Tailed Test