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. 2017 Jun 27;45(15):8930–8942. doi: 10.1093/nar/gkx560

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© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.

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Figure 5. — Selective usage of PAS within PTEN 3′UTR by Star-PAP controls cellular PTEN levels in response to DNA damage. (A) PAS distribution along the PTEN mRNA 3′UTR and the expression changes of the detected PASs after KD of the nuclear PAPs. PAS IDs and the location of the PTEN siRNA target site were indicated. (B) PTEN protein level changes after knocking down the individual PAPs in HEK293 cells. (C) Knockdown and rescue assay. No rescue of the PTEN protein expression was discerned with PAPα and Star-PAP^pd/sm ectopic expression after Star-PAP KD. In contrast, Star-PAP^wt/sm expression restored PTEN levels. (D) PTEN mRNA and protein levels in the cells with or without Star-PAP KD and in the presence or absence of DNA damage induction by etoposide treatment were examined by qRT-PCR (upper panel) and IB (lower panels). DMSO was used as vehicle control for the treatment. qRT-PCR primer pairs indicated with red arrows in (A) are located in the CDS region of the PTEN gene. (E) PTEN mRNA 3′UTR-specific KD reduced both basal and DNA damage-induced PTEN expression as measured by qRT-PCR for mRNA (upper panel) and IB for protein (lower panel) levels. The siRNA targeting site within the PTEN mRNA 3′UTR between PAS3 and PAS4 is shown in (A). (F) Analysis of PTEN 3′UTR expression by luciferase reporter assays. The 3′UTR of PTEN or GAPDH mRNA was subcloned into the pLightSwitch_3′UTR reporter vector for expression assessment in HEK293 cells using ELISA. The relative luciferase activities were normalized to the mock treatment control. Error bars represent standard error of the means of three independent experiments with triplicates for each experimental condition. Prom = promoter; MCS = multiple cloning site; DS = downstream sequence. The Error bars in (D), (E) and (F) represent mean ± s.d. of three independent experiments with triplicates for each experimental condition. **P < 0.001: significance relative to the control with one-way analysis of variance (one-way ANOVA).