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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 4. — Star-PAP regulates APA of select protein-coding and lncRNA genes. (A) 3′READS detected marked changes in the EIF4A1 mRNA isoform expression corresponding to the distal PAS after siRNA knockdown of Star-PAP, PAPα, or PAPγ. The PAP regulation of the expression of the EIF4A1 mRNA isoform generated using the distal PAS (B) and the protein expression (C) was evaluated using the knockdown and rescue experiments in HEK293 cells. (D) Luciferase reporter assays with the EIF4A1 distal PAS-flanking sequences that carry the wt, mutant AUA (Mut1) or mutant AUA and AAUAAA (Mut2). The bottom panel depicts the luciferase reporter constructs that were integrated into the pLightSwitch-3′UTR vector. (E) The effect of the PAP knockdowns on the usage of the NEAT1 RNA PASs was detected by the 3′READS. (F) Star-PAP-specific regulation of the distal PAS usage was confirmed by qRT-PCR. (G) Luciferase reporter assays with the NEAT1 distal PAS-flanking sequences that carry the wt, mutant AUA (Mut1) or mutant AUA and AAUAAA (Mut2). The luciferase reporter constructs were illustrated in the lower panel of (D). The primers for the relative expression of the mRNA isoform containing the distal PAS over the reference sequence of the EIF4A1 and NEAT1 RNAS were indicated by red and blue arrows, respectively, in (A) and (E). Error bars represent mean ± s.d. of three independent experiments with triplicates for each experimental condition. *P < 0.01 and **P < 0.001 represent the significance of the mean of three independent experiments relative to the control, and ^#P < 0.01 and ^##P < 0.001 were for the significance of the mean of three independent experiments relative to the siStar-PAP of the wt group based on one-way analysis of variance (One-way ANOVA) using StatPlus (AnalystSoft).