Fig 2: Working model figure describing splicing events of TERT observed in this study.A) Alternative splicing of TERT pre-mRNA produces splice variants. Only full-length TERT with all 16 exons can be translated into active telomerase. When exons are spliced out (e.g., exclusion of exons 7–8 in red box), transcripts are degraded or form inactive telomerase. B) TERT expression and telomerase activity in three different contexts (iPSC differentiation into NPC, increase in iPSC density, and Calu-6 cell density). Increase (red arrow) or decrease (blue arrow) or no change (—) are indicated for total TERT transcripts (FL TERT (7/8 included) plus minus beta TERT (7/8 excluded); ?+?), absolute amount of FL TERT transcript (7/8 included, ?), percentage of FL TERT transcripts compared to total (?/(?+?)), and telomerase activity. C) (Left panel) TERT AS regulators in iPSC differentiation into NPC or in calu-6 cells. PTBP1 promotes FL TERT in iPSCs whereas NOVA1 and PTBP2 promote minus beta TERT in NPCs. (Right panel) Three splicing factors were selected as cell type specific FL TERT-promoting candidates (HNRNPM in iPSCs; SRSF2 in both iPSCs and cancer cells; U2AF2 in cancer cells). TERT AS in Calu-6 cells was not affected by HNRNPM knockdown. On the other hand, SRSF2 or U2AF2 knockdown significantly reduced the amount and percentage of FL TERT (7/8 included). This figure was created with BioRender.com.

Fig 3: Knockdown of candidate splicing factors (SFs) in cancer cells (Calu-6) resulted in expected shifts in TERT splice variant expression.A-C) Knockdown of HNRNPM (A), SRSF2 (B), and U2AF2 (C) was confirmed by western blot. Representative images of selected SFs and beta-actin (loading control) (top panel). Knockdown was quantified by normalization with beta-actin then expressed relative to siRNA control (n = 6 biological replicates per condition; bottom panel). D) TERT splice variant expression ratio of exons 7/8 inclusion (potential FL) to exons 7/8 exclusion (minus beta) was reduced by knockdown of SRSF2 and U2AF2, not HNRNPM (determined by ddPCR; n = 6 biological replicates per condition). Student’s t test set at P = 0.05 for significance compared with siRNA-treated conditions with siControl (A-C; **, P < 0.01; *** P < 0.001; **** P < 0.0001). One-way ANOVA with uncorrected Fisher’s LSD for post hoc comparisons of siRNA treatments were used to compare siRNA-treated conditions with siControl (D; ***, P < 0.001; ****, P < 0.0001). Data are presented as means ± standard deviations where applicable.