Figure 3—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 3—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 3—figure supplement 2.
Download figureOpen in new tabFigure 3—figure supplement 2. Cytoplasmic NMD-refractory transcripts.

(A) The relative abundance of the intron-retaining Alu transcripts of MCM3 and NUP133 were measured in cytoplasmic and nuclear RNA from cells depleted of hnRNPC (siC #1 and #2), or control cells (‘no si’, no siRNA; siCtrl, control oligonucleotide). Semi-quantitative RT-PCR analysis is averaged across independent biological replicates (MCM3: n = 5, NUP133: n = 2), error bars represent s.d.m. (B) RNA abundance of the introns flanking the Alu-exon in MCM3 was measured by quantitative RT-PCR. As control, the overall mRNA abundance was quantified by an exon-exon spanning primer, and the abundance of the Alu-exon free intron 11 was measured in parallel. Cytoplasmic and nuclear RNA of cells depleted of hnRNPC (siC #1 and #2), or control cells (no siRNA and control oligonucleotide), and normalised to abundance of abundant RNAs (using ΔΔCt, control RNAs were eIF4G and SDH mRNAs). Shown are the averages across independent replicates (n = 3–4), error bars represent s.d.m. (C) The relative abundance of the intron-retaining Alu transcript of GMPS 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 across independent biological replicates (n = 3), error bars represent s.d.m. (D) A representative image of absorption at 230 nm is shown for the polysome profiles used in Figure 3D and in (E). Fractions from the polysome sedimentation were pooled as indicated. Bioanalyzer traces run with 100 ng RNA of each pool are shown below, demonstrating that ‘free RNA’ contains very little rRNA. (E) The relative abundance of the NMD-refractory Alu-exons in TNPO3 and ZFX were measured in RNA fractions from polysome gradients as in Figure 3D.

DOI: http://dx.doi.org/10.7554/eLife.19545.012