(A) Depletion of hnRNPC in total RNA led to significant expression changes in 601 protein-coding genes (p-value < 0.01), of which 166 contain an Alu-exon. Boxplot showing the distribution of expression fold changes (log2) over control (unspecific control oligonucleotide) in cells depleted of hnRNPC (siC #1 and siC #2) and/or UPF1 (siUpf1) as indicated below. Differences between Alu-exon gene abundance across groups were tested by Kruskal-Wallis Rank Sum test (p-value < 2.2e-16), and pairwise comparisons within each condition were tested with a two-sided Wilcoxon Rank Sum test. * and *** indicate p-value < 0.05 and 0.001, respectively. (B) Boxplot showing the changes in exon abundance upon hnRNPC and/or UPF1 depletion as fold changes (log2) over control (unspecific control oligonucleotide), analysed by DEXSeq (Lykke-Andersen et al., 2000). Only alternative exons and Alu-exons in protein-coding genes were considered. Differences between Alu-exon abundance across groups were tested by Kruskal-Wallis Rank Sum test (p-value < 2.2e-16), and pairwise comparisons were corrected according to Siegel-Castellan. *** indicates p-value < 0.001. (C) Boxplot showing relationship between gene expression changes upon hnRNPC depletion and Alu-exon usage. Alu-exons were stratified according to their fold change (log2) in exon abundance in cells depleted of UPF1 and hnRNPC (siC #1), which should be the maximal achievable inclusion of each Alu-exon. Protein-coding genes with Alu-exons were filtered for a minimal RPKM of 1 (2809 genes in total). Significant anti-correlation between Alu-exon inclusion and Alu-exon gene expression levels was tested by Spearman correlation (indicated by ρ coefficient). (D) Depiction of the overlap of hnRNPC- and NMD-sensitive exons that are significantly regulated (adjusted p-value < 0.01) under the conditions in (B). Exon sets as in (B). Dashed lines visualise proportion of exons that are coordinately regulated by both hnRNPC and NMD. hnRNPC+ and NMD+ denote upregulation in hnRNPC-depleted and UPF1-depleted cells, respectively. All proportions are drawn to scale within each group. (E) Bar diagram showing the proportion of Alu-exons outside (grey hatching) and inside the coding sequence (CDS), with the latter being further subdivided into exons introducing a PTC (PTC+, black), without PTC but introducing a frame-shift (grey), or being in frame (PTC-, white) for the following Alu-exon categories: non-regulated (hnRNPC-/NMD-, n = 313), NMD-specific (hnRNPC-/NMD+, n = 72), shared-target (hnRNPC+/NMD+, n = 207), and hnRNPC-specific (hnRNPC+/NMD-, n = 465). (F) Density plot showing the distance of the PTC to the downstream splice site for NMD-sensitive (orange) and NMD-refractory (green) PTC+ Alu-exons. Only Alu-exons that were supported by junction-spanning reads on either side in our RNAseq data were taken into account. In Figure 2—figure supplement 1, the lack of functional NMD due to UPF1 depletion is validated by semi-quantitative and quantitative RT-PCR as well as by analysis of transcriptomic changes in RNAseq of UPF1-depleted cells. In Figure 2—figure supplement 2, we present semi-quantitative RT-PCR quantifications of individual Alu-exon transcripts compared to the Alu-exon free transcripts. This validates that Alu-exon transcripts are depleted from the cytoplasmic RNA pool by NMD.