Supplementary Figure 7: NMD recognizes and degrades transcripts bearing GC-rich 3’ UTRs

(A-B) Sequence composition of CUG repeat sequences in the 3’UTR contributes to NMD transcript recognition for degradation. (A) Schematic drawing of the GC-rich or AT-rich plasmids for expressions in C. elegans muscle cells. (B) Fluorescent microscopy images of strains expressing a GFP with a 300bp ‘artificial’ insert in their 3’UTR containing the following GC percentages: 31%, 32%, 60% and 70%. Also included are the control strains containing 3’UTR inserts cloned from A. thaliana (34%GC) and P. aeruginosa (66%GC). These strains are shown in a wt background and in the background of the following smg mutants: smg-1(5861), smg-2(qd101) and smg-6(r896). The ‘fluorescence’ observed in the 60%GC and 70%GC strains in a wild type background corresponded to the characteristic gut autofluorescence, and no GFP signal was observed in the body wall muscle cells of these animals. Images were taken of animals at the L4 stage. Bar, 100 μm. (C-D) Western blot analysis of UPF1 down-regulation (24h post-transfection) by siRNA pool of unaffected (C) and DM1 (D) fibroblast cells, using UPF1-specific antibody. Fibroblasts showed a decrease of 40% in UPF1 levels relative to cells transfected with scrambled siRNAs (mock cells) in both unaffected (C) as well as DM1 (D) cells. GAPDH levels were used for normalization across samples.