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. 2017 Mar;23(3):378–394. doi: 10.1261/rna.058040.116

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© 2017 Li et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 1. — A new quantitative method, distinct from alternative splicing assays, distinguishes NMD activity from alternative splicing and transcriptional regulation. (A) Schematics of the RT-qPCR method specifically detecting alternative splicing isoforms. The NMD isoform can be either the inclusion or exclusion isoform, while the other isoform is designated as the non-NMD isoform. The inclusion isoform is detected by primer F1 and isoform-specific primer R1. The exclusion isoform is detected by F2 and isoform-specific junction primer R2. Primers F3 and R3 are commonly used in alternative splicing assays to detect both inclusion and exclusion isoforms. (B) Conventional splicing assay to derive expression ratios of the Psd-95 NMD isoform relative to the non-NMD isoform in N2a cells depleted of UPF1 using two independent siRNAs or N2a cells overexpressing PTBP1. The ratios by themselves do not distinguish NMD regulation from alternative splicing regulation. Representative digital gel images are shown in the lower panel. A one-way ANOVA test was used to determine significant ratio changes between different samples, followed by a Tukey's multiple comparison test. N = 3. (C) RT-qPCR analysis of the Upf1 deficient cells as in B but using different primers specific to the Psd-95 non-NMD isoform and Psd-95 NMD (NPsd-95) isoform along with validation of the siUpf1 knockdown efficiency. (D) RT-qPCR analysis of the PTBP1-overexpressing cells as in B but using different primers specific to the Psd-95 isoforms described in C. Western blots (right panel) of the samples transfected with GFP and Flag-PTBP1 plasmids. Arrow: Flag-PTBP1; arrowheads: endogenous PTBP1 proteins that are down-regulated by PTBP1 overexpression. (E) Responses of the two Psd-95 isoforms to double knockdown of PTBP1 and PTBP2 in N2a cells assayed by RT-qPCR. Right panel shows the Western blots verifying the knockdown efficiencies. (F) RT-qPCR analysis of the two Psd-95 isoforms 4 h after 4 µg/µL actinomycin D treatment. (C–F) A two-way ANOVA test followed by Dunnett's multiple comparison tests was used to determine significant expression changes between samples. (*) P < 0.05. Error bars represent mean ± SEM.