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

Open accessCopyright infoDownload PDFDownload figures

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

Figure 2—figure supplement 2.

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 2.
Download figureOpen in new tabFigure 2—figure supplement 2. Expression analysis of NMD-sensitive and NMD-refractory Alu-exons.

The relative abundance of the Alu-exon transcript of six genes was measured by semi-quantitative RT-PCR in cytoplasmic RNA from control, hnRNPC- and UPF1-depleted cells. Shown are gel visualisations of capillary electrophoresis and quantification of average Alu-exon inclusion (n = 3), error bars represent s.d.m. The upper scheme illustrates the position of the Alu-exon relative to the flanking constitutive and alternative cassette exons (if present, ‘ACE’) and the position of the primers used for RT-PCR. (A) Quantification of the Alu-exon transcripts of the AGL, NUP133, MCM3 and HELLS genes. These four Alu-exon transcripts were found to be significantly upregulated by co-depletion of hnRNPC and UPF1 and are examples of shared-target Alu-exons. Note that the Alu element within intron nine of MCM3 produces two different Alu-exons with alternative 5’ splice sites. (B) Quantification of a second Alu-exon transcript in the MCM3 and HELLS genes. These two Alu-exon transcripts were found to be refractory to NMD based on their abundance in UPF1/hnRNPC co-depleted cells. The same was observed for the Alu-exons in TNPO3 and ZFX, which also did not change in gene expression upon hnRNPC depletion. These represent examples of hnRNPC-specific Alu-exons. For comparison, quantification of the Alu-exon transcripts of TNPO3 and ZFX in nuclear RNA are shown. To test for significant changes in Alu-exon transcript abundance, one-way ANOVA was performed on each dataset from cytoplasmic RNA. Multiple comparison correction was done according to Tukey’s HSD.

DOI: http://dx.doi.org/10.7554/eLife.19545.009