Figure 1—figure supplement 4. | 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 1—figure supplement 4.

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 1—figure supplement 4.
Download figureOpen in new tabFigure 1—figure supplement 4. Cytoplasmic depletion of Alu-exon genes is not caused by a defect in mRNA export upon hnRNPC depletion.

(A) Percentage of reads mapping to snRNAs, introns or exons are shown for each sample. Examined were RNAseq samples of cytoplasmic and nuclear RNA of cells transfected with siRNAs for hnRNPC (siC #1 and #2) or controls (no siRNA, control oligonucleotide). snRNA abundance served as internal reference. (B) Assessment of the cytoplasmic accumulation of the ERK-inducible genes FOS, EGR2 and NDRG1 in control and hnRNPC-depleted cells. Cells were treated with 100 nM 4-HT for 0, 30 or 60 min, and reads mapping to each gene after normalisation for library size (normalised ‘counts’) are shown as line chart. Controls and hnRNPC depletion experiments as in (A). Our time-course showed ERK-induced genes increased first in the nucleus and then with a time-delay in the cytoplasm, in line with a transcriptional burst in gene expression. 4-HT: 4-hydroxytamoxifen. (C) A scatter plot comparing the fold change (log2) of genes upon depletion of hnRNPC in cytoplasmic and nuclear RNA. To estimate expression fold changes, we compared cells depleted of hnRNPC (siC #1 and #2) against controls (no siRNA, control oligonucleotide) using DESeq (Anders and Huber, 2010). Alu-exon genes are marked in red, and the upper-left quadrant is labelled as a pattern considered as consistent with a defect in mRNA export. (D) The ratio of junction-spanning to intronic reads was calculated for each gene in cytoplasmic and nuclear RNA. We separated Alu-exon genes associated with and without significant intron retention (adjusted p-value < 0.01, see also Figure 3). No accumulation of unspliced RNA in the nucleus is observed for either group of genes. (E) The abundance of NUP133 protein was tested by semi-quantitative Western blot in cells depleted of hnRNPC for 48, 60 and 72 hr. NUP133 protein levels gradually decrease in hnRNPC-depleted cells. Other components of the nuclear pore are not affected as demonstrated by the mAb414 antibody, which recognises FGF repeats found in all inner nuclear pore proteins marked at the side. LaminB protein levels served as loading control.

DOI: http://dx.doi.org/10.7554/eLife.19545.006