Region 2b forms a stem–loop important for oocyte entry of oskar mRNA. (A) Schematic of the secondary structure of the reporter mRNAs analyzed in this panel and their localization capacity during early oogenesis. Red indicates stretches with point mutations. (B) Alignment of the OES region in 12 Drosophila species with sequenced genomes. Highlighted in blue are nucleotides with >90% similarity. The experimentally validated secondary structure of the D. melanogaster OES is depicted below: A parenthesis indicates a base-paired nucleotide; an asterisk indicates a single-stranded nucleotide. Note that the terminal stem is almost entirely conserved among all species. (C) Primary sequence of the wild-type oocyte entry signal and the mutations analyzed in this panel. (D–K) Stage 5–6 egg-chambers from wild-type flies expressing transgenic oskar 3′ UTR-region 2 reporter mRNAs fused to the EGFP open reading frame under the control of mat-α4-tub-Gal4. Reporter RNAs are detected by fluorescent in situ hybridization using an egfp-antisense probe. A star indicates the position of the oocyte nucleus. Bar, 30 μm. Reporter mRNAs were fused to: region 2 of the oskar 3′ UTR (D); region 2 with deletion of proximal stem of region 2b: Δ2b-p (E); region 2 with a deleted distal stem of region 2b: Δ2b-d (F); region 2 with a deletion of the terminal loop (G); region 2 with a mutated 5′ stem (H); region 2 with a mutated 3′ stem (I); region 2 with mutations in both the 5′ and 3′ stem such that complementarity is restored (J); region 2 in which the quality of the terminal stem is changed from AU-rich to GC-rich (K).
