Fig. 1: Bidirectional profile of intronic APA (alternative polyadenylation) events in response to changes in mTOR signaling in cells.

a A schematic representation of the profiling of intronic APA events using RNA-seq data from cell lines. The RefSeq and UCSC gene structures were merged, and the RNA-seq data were quantified based on the collected structures. The chi-square test was used to determine significant intronic APA events between the control and the case based on the truncation ratio (TR), which was calculated as follows: TR = [quantity of intronic APA transcript] / [quantity of total transcript (intronic APA + full-length)]. The intronic APA events with a chi-square test p < 0.05 and TR difference >0.1 were considered significant events. b Scatter plots of the TRs of genes with low and high mTOR expression in cells. The analyses included wild-type (WT; low mTOR) and Tsc1^-/- (high mTOR) MEFs as well as the breast cancer cell lines MCF7, BT549, and MDA-MB-361. Cells were treated with 100 nM Torin 1 for 24 h to inactivate mTOR signaling in these cells. c, d Examples of RNA-seq read alignments of genes showing enriched intronic APA events in high-mTOR-Tsc1^-/- MEFs and low-mTOR-WT MEFs, respectively. The alignments are color-coded to highlight the regions with intronic APA events. e Real-time quantitative PCR (RT‒qPCR) validation of genes showing dynamic intronic APA events upon changes in mTOR signaling in cells. The analyses included WT, Tsc1^-/-, and Tsc1^-/- cells treated with Torin 1 (100 nM, 24 h). The Y-axis scale is presented on a log scale. Four technical repeats were conducted, and Student’s t tests were performed for statistical analysis. The data are presented as the mean (SD). f Validation of protein expression of intronic APA mRNAs in WT and Tsc1^-/- MEFs. (Left panel) Full-length (FL) and truncated (TR) isoforms of SIN3B and AGAP3 were detected using western blotting. (Right panel) Confirmation of FL and TR isoforms using RNAi. RNAi-mediated knockdown of SIN3B and AGAP3 FL or TR isoforms in Tsc1^-/- MEFs. g A mass spectrometry approach, namely, selected reaction monitoring (SRM), was used to detect TR isoforms in the Tsc1^-/- proteome. A peptide for TR PDXDC1 is highlighted as an example, with the peptide sequence originating from exonized intron sequences in the intronic APA transcript highlighted in the yellow box. The gene structure of the intronic APA transcript of Pdxdc1 is also shown. Fragmented ion spectra for the C-terminus of PDXDC1 from Tsc1^-/- cell extracts and synthesized peptide are shown as an example.