Effects of RNA binding mutations on MBNL1 splicing activity. A. Schematic representation of MBNL1. Zinc fingers are shown in black, the C-terminus in purple. Amino acid positions of deletion boundaries mutants and ZF domain inactivating mutations are indicated. The 382 aa MBNL1 isoform lacks sequences corresponding to exons 7 and 9. B. Comparison of wild type MBNL-N and MBNL-N-M1234 UV crosslinking to RNA. Upper panel, Coomassie blue stained gel; lower panel UV crosslinking. RNA used for crosslinking encompassed Tpm1 exon 3 and both upstream and downstream MBNL elements. The identity of crosslinked MBNL (lanes 1-3) was established by immunoprecipitation with anti-MBNL1 (lane 9) but not anti-GST antibodies (lane 8). The asterisked band is a contaminant that was not immunoprecipitated by anti-MBNL1; it does not correspond to the higher molecular weight contaminant in the Coomassie stained samples of mutant protein (lanes 4–6). C. Effects of MBNL1 knockdown and overexpression upon Tpm1 splicing in HeLa cells. The cartoon depicts the ΔBP minigene; the U and D MBNL binding elements and P3 and DY pyrimidine tracts are indicated. The minigene was co-transfected with control (C2, lane 1) or MBNL1 siRNAs (lanes 2–10), and with wild-type MBNL1 (lane 10) or MBNL1 mutants in the indicated ZF domains (lanes 3–9). Values significantly different from FL wild type MBNL1: **, P < 0.01; *** P < 0.001; ns, not significant. Values for the M1234 mutant are statistically significant, but lack of protein expression (panel F) prevents meaningful conclusions from being drawn. D. Effects of MBNL1 overexpression upon Vldlr splicing in HeLa cells. The cartoon depicts the Vldlr minigene; the MBNL1 binding site is indicated by the yellow diamond. The minigene was transfected alone (lane 1), with wild-type MBNL1 (lane 9) or mutants in ZF domains (lanes 2–8). E. Western blot of MBNL1 in mock transfected, control(C2) or MBNL1 siRNA treated cells. F. Anti-FLAG western blot showing expression of MBNL1 constructs from panels C and D.