Table 1.
MblC-specific sequences are found in distantly related invertebrates.
Figure 1.
Genetic modifiers of mblC overexpression exhibit different interaction strengths.
(A–H) Scanning electron microscopy of eyes from Or-R (A), sev-Gal4 UAS-mblC/+ (B), and flies expressing mblC as in (B) and simultaneously heterozygous for Traf1GS2154 (C), jumuL70 (D), TrafEP578 (E), aret01284(F), th5 (G), and nonAf00870 (H). Suppressors of mblC overexpression (C, D) ameliorated ommatidial irregularity to diverse extent. Enhancers (E–H) reduced eye size and/or increased roughness, and sometimes led to fusion of the overlying lenses. Halving jumeaux dose also suppressed mblC overexpression in the wing disc. Stereomicroscope images of wings from Or-R (I), en-Gal4 UAS-mblC/+ (J), and en-Gal4 UAS-mblC/+; jumuL70/+ (K). Arrows in (J) point to ectopic vein material. Bar graph representing the percentage of flies, with the genotypes indicated, that show a type I, II or III vein phenotype, or any vein phenotype (total). Phenotypic classes were defined as: I, L4 and L5 become fused and both of them disappear; II, L4 and L5 fuse but at least a vein was recognizable; III, L4 and L5 fuse, but are both recognizable. Wing in (J) would score as type III.
Table 2.
Suppressors and enhancers of a mblC overexpression phenotype.
Figure 2.
Muscleblind proteins regulate troponin T alternative splicing in vivo and in cell culture.
(A) Drosophila troponin T splicing characterisation in wild type (OrR); mblE27/CyO, ubi-GFP; mblK7103/CyO, ubi-GFP; and the hypomorphic allelic combination mblE27/mblk7103 early pupae. Molecular weight of DNA marker bands is shown on the left. Exon composition compatible with product size is shown on the right. Rp49 is shown as control in RT-PCR. (B) Murine TnnT3 minigene was co-transfected into HEK293T cells along with plasmids expressing Drosophila Muscleblind and Bruno proteins. +F indicates presence of the foetal exon and –F its absence. (C) Bar graph representing the average intensity of +F (light grey) and -F (dark grey) bands, as percentage of total, in three replica experiments, except for co-transfection of MblB that could only be amplified once. Statistically significant differences from vector alone controls (GFP lane) are denoted by an asterisk (p-value<0.01). Error bars are standard deviations. Bruno proteins did not significantly modify minigene alternative splicing.
Figure 3.
mblC overexpression activates apoptosis in vivo, but not significantly in cell culture.
Confocal micrographs of third instar wing imaginal discs from en-Gal4 mblC/+ (A,C,E) and en-Gal4/+ controls (B,D) stained with an anti-Mbl (A), anti mammalian Caspase-3 antibody (B,C), or TUNEL assay (D,E). Wing imaginal discs of en-Gal4 UAS-mblC (C) flies show activation of executioner caspase-3 in cells over-expressing MblC (A) in the posterior compartment where the en-Gal4 driver is active. Despite the fact that MblC overexpression levels are similar in posterior pouch (A, bent arrow) and notum cells (A, arrowhead), caspase-3 is not detected activated in prospective notum cells (C). A TUNEL assay to detect DNA fragmentation that results from apoptosis signalling cascades reproduced the same pattern of apoptotic cells (D,E) detected by caspase-3 activation. (F) Bar graph representing the average number (from quadruplicates) of live cells 48 h after transfection of plasmids expressing the indicated Muscleblind protein isoforms. Overexpression of Muscleblind isoforms did not significantly reduce Drosophila S2 cell viability in cell culture conditions. Error bars are standard deviations. (G) Western blot of protein extracts from S2 cells transfected as in (F) with the indicated Muscleblind proteins and detected with an anti-Muscleblind antibody [47]. Lower molecular weight bands in lanes MblA and MblB are degradation products. MblD could not be detected by western blotting. Predicted molecular weights are: MblA, 22.65 kDa; MblB, 34.46 kDa; MblC 26.91 kDa.
Figure 4.
Mutation of a conserved FKRP motif reduces nuclear localization and enhances cell death-inducing activity of MblC.
(A) ClustalW multiple alignment of part of Drosophila MblC-specific sequence (dmel) with homologous sequences from C. elegans (cel), Anopheles (aga), Danio rerio (dre) mus musculus (mmu; Mbnl1) and humans (hsa; MBNL1). SUMOplot web server predicts the Drosophila FKRP and C. elegans MKRP sequences (boxed) as sumoylation target sites. A conserved lysine (asterisk) was mutated to isoleucine in MblCK202I. (B) COSM6 cells transfected with 1 µg of GFP-tagged MblC protein showed preferential nuclear localization and perinuclear aggregates that increased in number in MblCK202I. (B) HEK293T cells transfected with 300 ng of GFP-tagged MblC showed no perinuclear aggregates whereas MblCΚ202Ι still aggregated. Mutant MblC (green) co-localized with CUG ribonuclear foci (red) in the cell nucleus (blue) stained with DAPI (C). (D) TnnT3 minigene splicing assay in HEK293T cells. GFP-tagged MblC and MblCK202I promoted foetal exon exclusion to the same extent compared to transfection of the empty vector (p<0.01). (E) Drosophila S2 cell viability assay 48 h after transfection of normal and mutant MblC. MblCK202I significantly reduced the number of viable cells (y-axis) compared to transfection of vector alone (p<0.05).