Identification of domains in MBNL1 responsible for interaction with RNA foci using AP-FRET.

<p>(A) Schematic representation of FL-MBNL1 and its deletion mutants. The FL-MBNL1 has four zinc-finger motifs (black boxes), a linker region (grey) and a C′ terminal domain (stippled). Each construct contains an N′ terminal EGFP tag. (B) Western blot of transfected EGFP-fused FL-MBNL1 and its deletion mutants in HEK293Ts; (lower panel) blot probed with antibodies for EGFP and (upper panel) probed with Dynein. (C) E% distribution for the different ROIs. Similar E% distribution was seen for FL-MBNL1 (n = 31), Del-C′ (n = 33) and Del-Linker (n = 34); Del-ZF4-C′ (n = 30) resulted in a dramatic drop in FRET efficiency values whereas Del-N′ (n = 33) showed loss of interaction. The line on the graph represents the positive FRET threshold level. (D) AP-FRET analysis of deletion mutants versus FL-MBNL1 was performed with line averaging (Line Av = 2). Representative donor and acceptor pre-bleach and post-bleach images for each experiment are shown.