90 nucleotides between PY tract and 3′-splice site are required for MBNL1 autoregulation. A, a schematic representation of the non-canonical intron 4 of MBNL1 and sequence of the 90-nucleotide MBNL1 response element. Black hatch marks indicate instances of YGCY motifs on the schematic with thickness of the hatch mark indicating clustered YGCY motifs. YGCY motifs are highlighted in black on the sequence. The 90-nucleotide MBNL1 response element was deleted from the MBNL1 minigene (Δ90 construct). Shorter deletions within this response element are indicated under the sequence (Del 1–5 with Δ indicating the length of the deletion). B, splicing of Δ90 minigene in HeLa cells in the absence (lane 2) and presence of overexpressed MBNL1-eGFP (lane 3) where lane 1 shows the wild type splicing of the MBNL1 minigene. C, gel shift of the MBNL1 response element. Concentrations of the protein increase from left to right: 0, 0.026, 0.2, 0.9, 6, 30, 200, and 1200 nm. D, splicing of Del 1–5 in HeLa cells in the absence (first of set) and presence (second of set) of overexpressed MBNL1-eGFP. Lane 1 shows the wild type splicing of the MBNL1 minigene, and lanes 2–11 are sets of the following deletions: Del 1, lanes 2 and 3; Del 2, lanes 4 and 5; Del 3, lanes 6 and 7; Del 4, lanes 8 and 9; and Del 5, lanes 10 and 11. nts, nucleotides.