Figure 2—figure supplement 1. | Splicing repression allows the gradual emergence of new Alu-exons in primate evolution

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Splicing repression allows the gradual emergence of new Alu-exons in primate evolution

Figure 2—figure supplement 1.

Affiliation details

UCL Institute of Neurology, United Kingdom; MRC-Laboratory of Molecular Biology, United Kingdom; Institute de Biologie de l’ENS (IBENS), CNRS UMR 8197, France; University College London Genetics Institute, United Kingdom; Goethe University Frankfurt, Germany; Institute of Molecular Biology (IMB), Germany
Figure 2—figure supplement 1.
Download figureOpen in new tabFigure 2—figure supplement 1. Validation of disenabling NMD by UPF1 depletion.

(A) Quantitative RT-PCR was used to measure the abundance of three genes known to be NMD substrates, GADD45B, SMG5 and ATF3 (Chan et al., 2007), as well as HNRNPC and UPF1 for control. Each of the NMD targets was upregulated to similar extent in UPF1-depleted or UPF1 and hnRNPC co-depleted cells (n = 2–3). Error bars represent s.d.m. (B) Depletion of UPF1 led to stabilisation of a known NMD-sensitive isoform of SC35 specifically in the cytoplasmic fraction, measured by semi-quantitative RT-PCR. Data are plotted as average of three independent replicates, error bars represent s.d.m. (C) The efficiency of UPF1 and hnRNPC depletion and co-depletion was tested by semi-quantitative Western blot. (D) To test our PTC prediction, we examined the change in expression of exons with or without PTC, comparing UPF1-depleted cells to control. As exon set, we used all UCSC-annotated alternative exons in protein-coding genes. (E) Comparison of the number of Alu-exons that we were able to annotate from our previously published work in HeLa cells (Zarnack et al., 2013) and the RNAseq data collected in HR1 cells (including UPF1/hnRNPC co-depleted cells). (F) The distribution of distances of the PTC to the downstream splice site is presented for NMD-sensitive and NMD-refractory exons. The analyses are based on all significantly regulated alternative PTC+ exons with junction-spanning reads on both splice sites.

DOI: http://dx.doi.org/10.7554/eLife.19545.008