Fig 1: 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); ➀+➁), absolute amount of FL TERT transcript (7/8 included, ➀), percentage of FL TERT transcripts compared to total (➀/(➀+➁)), 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 2: 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.
Fig 3: Impact of iPSC cell density on SF expression of TERT AS candidate SFs.A-F) Western blots of splicing factors and correlation analyses in different iPSC density. Top are representative images and bottom are scatter plots showing correlations between housekeeping gene-normalized SF expression levels and percentage of TERT exons 7/8 inclusion (n = 4 biological replicates per condition). Antibodies targeting HNRNPA2B1 (A), HNRNPCL1 (B), HNRNPH (C), HNRNPM (D), SRSF2 (E), and SRPK1 (F) were used for the western blots. 95% Confidence bands, Pearson’s correlation coefficient (r) and p values are shown. For the correlation analysis, 24 data points are included (six conditions x four replicates).
Fig 4: Analysis of TERT AS minigene loss-of-function screen and patient tumor expression profiles of splicing factors identifies top candidate SFs related to TERT splicing in lung cancer cells.A) TERT minigene data of selected SFs showing effect of SF knockdown on TERT exons 7/8 splicing in HeLa cells [16]. B-F) Log2-transformed RSEM values of SFs gene-expression levels (n = 58 matched LUAD patient samples). SRSF2 (B), U2AF2 (C), HNRNPA2B1 (D), SRPK1 (E), and HNRNPA1 (F) are significantly upregulated in tumor tissues from LUAD patients compared to tumor-adjacent normal tissues. Paired Student’s t test set at P ≤ 0.05 for significance compared with normal tissue controls (B-F; ** P < 0.01; *** P < 0.001; **** P < 0.0001). In the box plots, the lower boundary of the box indicates the 25th percentile, a line within the box marks the median and the higher boundary of the box indicates the 75th percentile. Whiskers above and below the box indicate the 10th and 90th percentiles. Points above and below the whiskers indicate data outside the 10th and 90th percentiles.
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