Figure 6

Comparison of MBNL1 deletions and point mutations upon tethered repression and activation. A. Schematic of the full-length MBNL1-MS2 construct with boundaries of various deletions indicated. B. Anti-MS2 western blot of proteins used in subsequent panels. C. The Tpm1 ΔBP DMS2 minigene was transfected alone (lane 1), or co-transfected with MS2 (lane 17), MS2 fused to full-length MBNL1 (lane 2), or the various deletion constructs indicated by the amino acid coordinates. Δ116-183 (lane 4) is a natural MBNL1 isoform resulting from exon skipping. Lanes 14–16 show the effects of the ZF1 and 2 mutations in the context of the 2-116-MS2 deletion mutant. In the experiment shown, the 2-253-MS2 was not expressed (lane 3); in other experiments (e.g. Figure 2E) its activity was similar to FL-MBNL1. The asterisked band is an artefactual band that does not appear in most experiments. Values significantly different from wild type MBNL1-FL-MS2 in panels C and D: **, P < 0.01; *** P < 0.001; ns, not significant. D. The Vldlr-MS2 minigene was co-transfected with the same effectors as panel C. E. Comparison of effects of mutations in panels C and D. The histogram indicates “% activity” for each of the MBNL1 constructs with the Tpm1 (green bars) and Vldlr (red bars) substrates. The values shown are relative to the activity of FL-MBNL1-MS2. 100% activity is defined by the difference in exon skipping/inclusion in the presence of FL-MBNL1-MS2 (upper horizontal line in histograms of panels C and D) and in the absence of co-transfection (lower horizontal lines). The comparison shows more rapid decline of activation than repression with C-terminal deletions into the ZF23 linker.