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. 2021 Jun 25;12:3965. doi: 10.1038/s41467-021-24046-3

Fig. 2. SMG7 requires interaction with SMG5 to rescue the SMG7 knockout phenotype.

Fig. 2

a Schematic representation of the SMG7 domain structure on the left. The proposed functions of the domains are indicated and mutated constructs and their expected effect are shown below. The illustration on the right depicts which mutation is expected to impair which individual SMG7 function and/or protein-protein interaction. b Model of the SMG5-SMG7 heterodimer structure. Human SMG7 (PDB ID: 1YA0) was modeled on the C. elegans SMG5-SMG7 structure (PDB ID: 3ZHE). Critical SMG7 mutations are highlighted in red and the indicated sulfate ion mimics a phosphorylated residue. c Western blot after FLAG co-immunoprecipitation (IP) of FLAG-tagged GST (control) or SMG7 constructs in SMG7 KO cells (n = 1). Tubulin serves as a control. d Quantitative RT-PCR-based detection (qPCR) of SRSF2 isoforms and ZFAS1 was carried out in the indicated cell lines upon expression of the indicated FLAG-tagged rescue constructs. The ratio of NMD isoform to canonical isoform (SRSF2) and ZFAS1 to the C1orf43 reference was calculated; data points and means from the qPCRs are plotted as log2 fold change (log2FC) (n = 3 biologically independent samples). Rescue efficiency was calculated based on the mean log2FC in relation lane 1 (set to 1) and lane 2 (set to 0). Western blot analyses are shown below (n = 1). Tubulin serves as a control. e Heatmap of quantitative RT-PCR-based detection (qPCR) of SRSF2 isoforms and ZFAS1 in the indicated cell lines upon treatment with the indicated siRNA. The ratio of NMD isoform to canonical isoform (SRSF2) and ZFAS1 to the C1orf43 reference was calculated; mean log2 fold change (log2FC) is shown (n = 3 biologically independent samples). The corresponding individual data points are plotted in Extended Data Fig. 3b. f Heatmap of quantitative RT-PCR-based detection (qPCR) of SRSF2 isoforms in the indicated cell lines upon treatment with the indicated siRNA and expression of the indicated FLAG-tagged rescue constructs. The ratio of NMD isoform to canonical isoform (SRSF2) was calculated; mean log2 fold change (log2FC) is shown (n = 3 biologically independent samples). The corresponding individual data points are plotted in Extended Data Fig. 3c. Clustering (k = 3) and functional summary of SMG7 mutations for NMD activity are depicted below.