Figure 1—figure supplement 2. | 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 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 1—figure supplement 2.
Download figureOpen in new tabFigure 1—figure supplement 2. Quality control of cytoplasmic and nuclear RNA fractions.

(A) The efficiency of hnRNPC depletion was tested by semi-quantitative Western blot. For comparison, a dilution series of lysate from control cells was included. (B) Cytoplasmic and nuclear protein lysates were collected together with RNA lysates, and probed for marker proteins by Western blot. GAPDH and H2A were chosen as abundant cytoplasmic and nuclear marker proteins, respectively. No cross-contamination of the cytoplasmic lysate with nuclear lysate was detectable, nor vice versa. (C) Quantitative RT-PCR was used to measure the abundance of a representative set of RNAs, in cytoplasmic and nuclear RNA or control reactions without template cDNA (NTC). Top: 5S rRNA and processed U1 snRNA were used as proxy for total RNA abundance. Unprocessed U1 snRNA is expected to be more abundant in the nucleus than in the cytoplasm (pre-U1). Bottom: To test for leakage of intronic sequences into the cytoplasm, two abundant mRNAs (GAPDH, HNRNPC) were quantified by two distinct sets of primers amplifying either an exon-exon junction or an intronic sequence. Intronic RNA was readily amplified from nuclear RNA but was close to negative control levels in which the RT enzyme was omitted (‘-RT’) in cytoplasmic RNA. This indicated that leakage of intronic RNA due to disruption of nuclei was negligible. (D) RNAseq data of cytoplasmic and nuclear RNA of HR1 cells depleted of hnRNPC was used to identify genes differentially expressed upon hnRNPC depletion, using DESeq (Anders and Huber, 2010). For each group, enrichment for genes containing Alu-exons was tested against all expressed genes using Fisher’s exact test. Enrichment of Alu-exon genes among genes with expression changes in cytoplasmic RNA explain the enrichment within all differentially expressed genes. ‘DiffExpr genes’ are all genes with padj < 0.01, combining differentially expressed genes from the cytoplasm and nucleus.