Figure S6.

hnRNP C Suppresses the Exonization of an Alu Element in the NUP133 Gene through Competition with U2AF65, Related to Figure 5

(A) Genome browser view including an Alu element (orange) plus the downstream exon within the NUP133 gene on chromosome 1 (229,601,700-229,601,100). hnRNP C iCLIP data (blue) show crosslinking at the upstream and the linker U-tract of the Alu element (corresponding thymidines are highlighted in red at the bottom). Little U2AF65 crosslinking (purple) is detected at the U-tracts in control HeLa cells, while strong crosslinking is observed in the HNRNPC knockdowns (KD1 and KD2). RNA-seq data (green) show exonization of the Alu element in both knockdowns, giving rise to three different Alu exon variants. The corresponding isoforms are schematically indicated: Alu suppression in isoform S, Alu inclusion as a cassette exon in isoform 1 and 2 (differing in the usage of an upstream or downstream 5′ splice site, respectively) and Alu inclusion using an alternative 3‘ splice site for the downstream exon (isoform 3). The wild-type (WT) sequence surrounding the 3′ and 5′ splice sites (arrowheads) including the introduced point mutations (3^mut and 5^mut) are shown below. The positions of the point mutations are highlighted in the sequences by black squares.

(B) U-to-C transitions promote Alu exonization in the presence of hnRNP C. Shown is the gel-like view of capillary electrophoresis of RT-PCR analysis of minigenes containing the Alu element in the NUP133 gene described in (A) with two flanking exons. The wild-type (WT) minigene shows no Alu exonization in control HeLa cells (Ctrl) and a significant increase in isoforms 1-3 in the HNRNPC knockdown (KD1 and KD2). A mutant minigene containing three T-to-C transitions in the upstream U-tract (3^mut) shows significant inclusion of isoforms 1-3 in control HeLa cells. Additional T-to-C transitions in the linker U-tract (5^mut) further elevate the inclusion of isoforms 1-3 in control HeLa cells and prevent any further regulation by hnRNP C (KD1 and KD2). The sizes corresponding to the different analyzed isoforms are schematically indicated on the right.

(C) Average Alu exon inclusion in percent for three replicate RT-PCR experiments as described in (B). The different isoforms are indicated by shades of gray (light gray: isoform 1; medium gray: isoform 2; dark gray: isoform 3). Lines indicate relevant comparisons with asterisks indicating different levels of significance for changes in the summed inclusion isoforms (^∗: p value < 0.05; ^∗∗: < 10⁻³; n.s.: not significant; Student’s t test). Error bars represent SD of the mean; n=3.