Supplementary Figure 2: Characterisation of the 3Mir Mutant

SUPPLEMENTAL DATA

Supplementary Figure 1: Generation and characterisation of a deficiency covering the cluster of microRNAs by Ends-Out Homologous Recombination. The dGMAP gene containing the microRNA cluster is shown in the upper panel. RH3miR represents the construct used for homologous recombination. Black boxes represent sequences homologous to the dGMAP locus. Dashed sequence is specific to the vector used. 3miR is a schematic drawing of the genomic locus after homologous recombination between the RH3miR construct and the dGMAP locus. The 3miR line consists of the replacement of the intronic sequence of the dGMAP locus (covering the three microRNAs) by the white gene sequence. Three pairs of primers were used to test the 3miR line by PCR, as shown below the drawings. Each PCR fragment was sequenced to confirm the breakpoints. Note that the 5’ anti and 3’ sense primers are not present in the genome of the white strain used for homologous recombination.

Supplementary Figure 2: Characterisation of the 3miR mutant.

Nuclei are visualized by DAPI staining (A,D,G, J’ and K’), membrane by phalloidin staining (B, J and K), and dGMAP (E, H, J” and K”), Wg (F and I) and Golgi (C) by immunofluorescence in 3miR (A-C, G-I and K-K”) or wt (D-F and J-J”) embryos (A-I) or egg chambers (J-K). Note that the dGMAP level strongly decreases in 3miR embryos (H) and egg chambers (K”). Note also that majority of 3miR embryos die before cellularisation stage (nuclei are observed (A) without membrane formation (B)) whereas 3miR escapers present wt organization (G, I and K).

Supplementary Figure 3: Summary of the rescue experiments.

3miR/FM7; arm-GAL4 females were crossed to males with homozygote insertions of different UAS downstream of which the expression of dGMAP, or individual microRNA or EGFP (as control line) can be induced by a GAL4 driver. The percentage of 3miR male survivors obtained in each cross was calculated and compared to the EGFP control cross. The 3miR semi lethality was rescued by 17-25% when one of the microRNA was expressed, but not upon expression of dGMAP. Between 55 and 120 3miR males were counted for each rescue experiment.