The Role of Oocyte Transcription, the 5′UTR, and Translation Repression and Derepression in Drosophila gurken mRNA and Protein Localization  Carol Saunders, Robert S Cohen  Molecular Cell  Volume 3, Issue 1, Pages 43-54 (January 1999) DOI: 10.1016/S1097-2765(00)80173-2

Figure 1 grk mRNA Is Produced in the Oocyte (A) Whole-mount in situ hybridization to gpro-Z transcripts. Transcripts are detected in the oocytes (arrowheads) but are not localized. (B and C) Whole-mount in situ hybridization to endogenous grk (B) and osk (C) transcripts in sister ovarioles of a colchicine-fed fly. The grk transcripts are concentrated in the oocyte, while the osk transcripts are concentrated in nurse cells. (D) Whole-mount in situ hybridization to endogenous grk transcripts in egg chambers of a cytochalasin D–fed fly. Molecular Cell 1999 3, 43-54DOI: (10.1016/S1097-2765(00)80173-2)

Figure 2 Localization Dynamics of grk mRNA in Colchicine-Treated and Untreated Oocytes Oocytes were doubly labeled for grk mRNA (green) and nuclear lamin (red). The oocyte nuclei are intensely stained and appear in the center of the micrographs. Follicle cell nuclei are small and at the periphery of the micrographs, while nurse cell nuclei are large and to the anterior (left) of the oocyte. (A) Various stage oocytes from colchicine-fed flies. Note that the mRNA is aggregated into one or a few clumps along a single side of the nucleus. (B) Untreated oocytes of the following stages: i, 6; ii, 7; iii, 8; and iv, 9. Panel ii shows a dorsal-midline view, while the other panels show lateral views with dorsal on top. (C) A sagittal series of optical sections through a single stage 7 oocyte undergoing nuclear migration. A lateral view (dorsal on top) of the reconstructed image is shown at the bottom right. Molecular Cell 1999 3, 43-54DOI: (10.1016/S1097-2765(00)80173-2)

Figure 7 The Role of K10 and the 3′UTR in grk Translation (A and B) Localization of grk mRNA (green) and protein (red) in wild-type (A) and K10 mutant (B) oocytes. Stages: (i) early stage 7; (ii) late stage 7/early stage 8; (iii) late stage 8; (iv) stage 9. In wild-type late stage 7/early stage 8 oocytes, Grk (arrowheads) is restricted to the anterodorsal corner of cell, even though grk mRNA is found all along the anterior cortex. In contrast, Grk accumulates all along the anterior cortex of the late stage 7/early stage 8 oocytes of K10 mutants. (B, iv) Control showing that K10 gene activity is not required for the association of grk mRNA (green) with the oocyte nucleus (red). (C and D) Immunolocalization of β-gal fusion protein produced by the gz3′ (C) and gzΔ3′ (D) transgenes. Stages: i, stage 6; ii, stage7/8; iii, stage 9/10. Note that fusion protein (arrowheads) accumulates all along the anterior cortex of gzΔ3′ transformants but is restricted to the anterodorsal corner of gz3′ transformants. The high background staining seen throughout the ooplasm of gzΔ3′ and gz3′ transformants is likely due to inefficient turnover of fusion protein synthesized during earlier stages of oogenesis, since the intensity of the such staining peaks during stage 6 and gradually disappears thereafter. Molecular Cell 1999 3, 43-54DOI: (10.1016/S1097-2765(00)80173-2)

Figure 3 Localization Dynamics of the ER and grk mRNA during Oogenesis Stage 5 (A–C), early stage 7 (D–F), and stage 9 (G–I) oocytes doubly labeled for the ER (A, D, and G) and grk mRNA (B, E, and H). (C, F, and I) Merged images. Arrowheads mark that side of the nucleus that lies farthest from the overlying follicle cell epithelium. Molecular Cell 1999 3, 43-54DOI: (10.1016/S1097-2765(00)80173-2)

Figure 4 Efficient grk mRNA Localization Requires the grk 3′UTR, but Not Translation of the Signal Sequence Whole-mount in situ hybridization to gzΔ3′ (A) and gz3′ (B) transcripts. Note the more rapid disappearance of gz3′ transcripts from the anterior cortex (compare the two middle panels). (C) Whole-mount in situ hybridization to gzstop transcripts, which contain a stop codon at the beginning of the grk protein-coding sequence (see text). (D) Immunolocalization of gzstop- encoded protein, with an anti-β-gal antibody. No protein is detectable, indicating that the stop codon has effectively blocked translation. Molecular Cell 1999 3, 43-54DOI: (10.1016/S1097-2765(00)80173-2)

Figure 5 Low-Resolution Deletion Analysis of the grk 5′UTR The starting construct (gz3′) is shown at the top of the diagram (see Experimental Procedures). The AB, BC, and B diagrams highlight the nondeleted portions of the ABgz, BCgz, and Bgz constructs, respectively (for details, see Experimental Procedures). The bottom panels show representative whole-mount in situ hybridization to ABgz and Bgz transcripts, respectively. The ABgz transcripts exhibit a normal localization pattern, while only diffuse distribution throughout the ooplasm is seen for Bgz transcripts. The distribution pattern of BCgz transcripts (not shown) is indistinguishable from that of Bgz transcripts. Molecular Cell 1999 3, 43-54DOI: (10.1016/S1097-2765(00)80173-2)

Figure 6 Localization and Translational Control Elements in the grk 5′ UTR (A) Schematic diagram and localization summary of linker scan mutations in the 5′UTR. The mutations are identified by the nucleotides substituted, for example, the 36/65 mutation carries a substitution of nucleotides +36 to +65, where +1 corresponds to the first nucleotide of the mRNA. All of the substitution mutations were derived from the gz 3′ construct (diagrammed at the top of the figure and see Experimental Procedures). The Δ36/347 mutation is a deletion of the segment +36 to +347, where the translation start codon begins at +355. This mutation was made with and without the 3′UTR (see Results and Experimental Procedures). The grk 5′ intron, which follows residue +164 (see Experimental Procedures), is not shown for simplicity. Early localization refers to posterior localization and the cortical arc and anterior ring transition stages (see Results). Late localization refers to anterodorsal localization. All transcripts that failed to exhibit wild-type anterodorsal localization were degraded during late stage 8 and 9. (B) Representative in situ hybridization to transcripts of the large deletion (Δ36/347z3′) that contains the 3′UTR. (C) Immunolocalization of lacZ fusion protein in oocytes carrying the gz3′ (left panel) and Δ36/347z3′ transgenes, which contain and lack GLE2, respectively. Molecular Cell 1999 3, 43-54DOI: (10.1016/S1097-2765(00)80173-2)