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. 2020 Mar 1;34(5-6):413–427. doi: 10.1101/gad.332270.119

Figure 3.

Figure 3.

SRSF2 promotes NMD when tethered downstream from a PTC, an effect that is dependent on splicing. (A) Diagram of cDNA constructs and Western blotting (right) of HeLa cells transfected with the indicated cDNAs using anti-T7-tagged and anti-β-tubulin (tubulin) antibodies. Note that MS2 cDNA does not have a T7 tag. (B) Diagram (not to scale) of HBB reporters with MS2-binding sites formed after splicing at the first or second exon–exon junctions, respectively (E1/E2-MS2 and E2/E3-MS2, respectively). Note that the binding motifs for both SRSF2 WT (GGWG) or SRSF2 mutant (CCWG) in exon 2 were inactivated to AAWG motifs (W = A/U). (C) Radioactive RT-PCR of the HBB reporters shown in B in HeLa cells cotransfected with the indicated cDNAs. (D) mRNA bands in C were quantified, normalized to GFP, and plotted as T39/WT (%) (mean ± SD, n = 3). (**) P < 0.01; (NS) not significant, t-test. (E) Diagram (not to scale) of HBB E2/E3-MS2 reporters shown in B, with sequential deletions of introns. (F) Radioactive RT-PCR of the HBB reporters shown in E in HeLa cells cotransfected with the indicated cDNAs. (G) mRNA bands in F were quantified, normalized to GFP, and plotted as T39/WT (%) (mean ± SD, n = 3). (*) P < 0.05; (NS) not significant, t-test. An intron 1 retained transcript variant is marked by a hash (#) in C. (TC) Normal termination codon; (PTC) premature termination codon.