Fig. 7: eIF6 missense mutations fully rescue the larval lethality of Sbds-deficient D. melanogaster.

a–c Cytoplasmic localization of Drosophila Sbds by (a) immunostaining of FLAG-tagged Sbds (red) in ovarian follicle cells, the nucleus in blue (DAPI), scale bar: 10 μm, 3 replicates, n = 30; (b) immunoblotting of third instar Drosophila larval cytoplasmic (C), soluble nuclear (N) and insoluble nuclear (I) fractions (3 replicates); (c) indirect immunofluorescence of the third instar larval wing disc cells. Sbds (red) depleted by RNAi in posterior wing disc cells (marked with GFP); the nucleus is blue (DAPI), scale bar: 10 μm, 3 replicates, n = 30. d RNAi depletion of Sbds in third instar larval extracts revealed by immunoblotting (3 replicates). e Sbds is required for cellular growth. RNAi depletion of Sbds in developing wings versus control. Wing size (n = 15, p-value <0.0001, left) and bristle density (n = 10, p-value <0.0001, right) as a percentage (±s.e.) of control. Scale bar: 200 μm. A two-tailed student t-test was used. f Drosophila Sbds (CG8549) locus. PiggyBac-element insertion site (arrow) and Sbds coding region (magenta) are shown. g Indicated proteins revealed by immunoblotting of larval extracts from indicated genotypes (3 replicates). h eIF6-N106S mutation or eIF6 dose reduction rescues larval lethality of Sbds-deficient flies. Development at indicated time-points after egg laying is shown. Scale bar: 1 mm. i Genetic complementation of homozygous Sbds^P/P mutant flies (at least 4 replicates, minimum n = 156; error bars represent mean ± s.e). j SDS-related eIF6 mutant protein expression in WT larvae expressing eIF6 WT or missense mutants (3 replicates). k eIF6-N106S and R61L variants have lower affinity for the 60 S subunit. Larval extracts were fractionated by sucrose gradient sedimentation and proteins visualized by immunoblotting (3 replicates). l EIF6-N106S rescues the cytoplasmic redistribution of eIF6 in Sbds-deficient flies. Subcellular fractions of third instar larvae cells with the denoted genotypes were immunoblotted to visualize the indicated proteins (3 replicates). m Subcellular distribution of endogenous eIF6 in the denoted genotypes quantified by densitometry of (l). Error bars represent mean ± s.e.; 3 replicates. Drosophila strains and genotypes are listed in Supplementary Tables S2a, b.