Figure 1.

MID1 binds to CAG repeat RNAs irrespective of the repeat-flanking sequences. (A) The binding of three different CAG repeat RNAs to MID1 was analyzed. Upper panel: schematic showing the predicted hairpin folding as well as the different CAG repeat flanking regions of ATXN2, ATXN3, and ATXN7 to visualize the different RNA fragments that were used, different colors symbolize the different flanking regions. Lower panel: in vitro transcribed fragments of ATXN2, ATXN3, and ATXN7 containing either normal or mutant CAG repeats were incubated with MID1-FLAG containing lysates. RNA-bound MID1 protein was analyzed on western blot using FLAG-antibodies. A representative western blot of n = 3 experiments is shown. (B) RNA-immunoprecipitation was performed in lysates from either mouse embryonic fibroblasts expressing mutant ATXN2 or primary neurons of a SCA3 mouse model that expresses full-length human ATXN3 with a mutant CAG repeat. MID1 was immunopurified and the presence of ATXN2 (upper panel) or human ATXN3 (lower panel) in the MID1 complex was tested by RT-PCR on RNA that co-purified with MID1. As negative control, a sample with unspecific IgG was used. As additional negative control a–RT reaction was performed. RT-PCR products were analyzed on an agarose gel. A representative gel of n = 3 experiments is shown.