Drosophila let-7 microRNA is required for remodeling of the neuromusculature during metamorphosis - PubMed (original) (raw)

Figure 1.

Drosophila let-7-C locus, knockouts, and rescuing transgenes. (A) The Drosophila let-7-C locus, located at cytological location 36E on chromosome 2, encodes a 2435-nt primary transcript containing three evolutionarily conserved miRNAs: miR-100, let-7, and miR-125. (B) The let-7-CKO1 and let-7-CGKI mutations contain 1071- and 991-base-pair (bp) deletions respectively, removing miR-100, let-7, and miR-125. The CG10283 gene, located proximally to let-7-C, is also disrupted by the let-7-CKO1 mutation. The let-7-CGKI mutation contains Gal4 coding sequences driven by let-7-C transcription (see Supplemental Material for details on let-7-CGKI strain generation). (C) The P{W8, let-7-C} rescuing transgene includes a 17,983-bp genomic fragment containing the let-7-C locus. Derivatives of P{let-7-C} (not shown) contain 10- to 15-bp deletions removing portions of the mature miR-100, let-7, or miR-125 sequence (see the Supplemental Material for details of P{W8, let-7-CΔmir-100}, P{W8, let-7-CΔlet-7}, and P{W8, let-7-CΔmiR-125} transgene construction). (D) Expression of miR-100, let-7, and/or miR-125 RNA is eliminated in the let-7-CKO1, let-7-CGKI, and let-7-CKO1/GKI strains and is restored by P{W8, let-7-C} and derivative rescuing transgenes. Samples of total RNA from 1-d-old male flies of the following genotypes were analyzed by Northern blot: wild type in lane 1, let-7-CKO1/KO1 in lane 2, let-7-CGKI/GKI in lane 3, let-7-CKO1/GKI in lane 4, let-7-CKO1/GKI; P{W8, let-7-C} in lane 5, let-7-CKO1/GKI;P{W8, let-7-CΔmir-100} in lane 6, let-7-CKO1/GKI;P{W8, let-7-CΔlet-7} in lane 7, let-7-CKO1/GKI;P{W8, let-7-CΔmiR-125} in lane 8, and P{UAS-let-7-C}; let-7-CKO1/GKI in lane 9. Northern blots were probed for miR-100, let-7, miR-125, and miR-1 RNAs, and miR-1 expression was used as a loading control.