Fig. 5

Evolutionary conservation of unstable, proline-rich U2af26 frames encoding PxxP motifs. a Evolutionary tree of mammalian species harboring a U2af26 gene generated with PhyloT. For each species the length of possible ORFs in all three hypothetical frames encoded by the last U2af26 exon are depicted; the annotated C-terminus of U2af26 is defined as frame 0. Potential ORFs with more than 50 AAs are highlighted in red. The asterisks mark frames accessible via an alternative 3′ss. b Frequency of SH3-domain-binding proline-rich PxxP motifs in extended − 1 or + 1 frames of U2af26 in comparison to the fl frame 0 (left panel). Proline content of extended U2af26 alternative frames compared to the UniProt/SwissProt average representing all proteins (right panel). c–e Stability of N-terminally GFP-tagged U2af26 frames from different species: protein stability of all possible rat (c) and human frames (d), including the frame accessible through an alternative 3′ss, and the alternative extended elephant frame − 1 (e). Stability was determined as in Fig. 3c (n > 3). (p < 0.001: ***, p < 0.01: **, p < 0.05: *). f, g Enrichment of interaction partners of full-length (f) and frameshift (g) isoforms of U2af26-GFP by quantitative mass spectrometry after GFP pulldown. Interaction partners are enriched with heavy/light (bait/bead control) intensity ratios, two independent experiments are plotted to show reproducibility. GFP-tagged U2af26 is shown in red; proteins known to be involved in splicing are shown in green and previously identified proteins that interact with or are part of the U2snRNP are shown in yellow. Two examples for specific interaction partners are shown