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Me31B silences translation of oocyte-localizing RNAs through the formation of cytoplasmic RNP complex during Drosophila oogenesis

Akira Nakamura^1,2, Reiko Amikura^1,*, Kazuko Hanyu¹ and Satoru Kobayashi^1,2,*,

¹ Institute of Biological Sciences, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
² Gene Experiment Center, University of Tsukuba, Tsukuba, Ibaraki 305-8572, Japan
^* Present address: Center for Integrative Bioscience, Okazaki National Research Institutes, Okazaki, Aichi 444-8585, Japan

View larger version (71K): [in a new window]   Fig. 1. Distribution of Me31B protein. (A) A confocal micrograph of stage 6 egg chamber expressing GFP-Me31B (green). The egg chamber is stained with phalloidin (red). GFP-Me31B signals show cytoplasmic particles in both nurse cells and the oocyte. (B) Higher magnification to highlight a ring canal. GFP-Me31B particles appear to pass through ring canal. (C-F) Distribution of endogenous Me31B. Wild-type ovaries were immunostained with an affinity-purified anti-Me31B antibody. Endogenous Me31B also distributed in a granular pattern in the cytoplasm of nurse cells and oocytes. Note that the antibody failed to permeate well in nurse cells in stage 10 egg chamber (F). (G) Cleavage embryos immunostained with anti-Me31B antibody. Me31B is uniformly distributed. By the cellular blastoderm stage (H), Me31B signal becomes undetectable. Scale bar in B: 5 µm.

View larger version (76K): [in a new window]   Fig. 2. Colocalization of Me31B with oocyte-localizing RNAs. Wild-type ovaries were double stained for Me31B (green) and osk (A-C), BicD (D-F) and vas (G-I) mRNAs (red). Higher magnification in the cytplasmic region of the nurse cells were also shown (insets). osk and BicD mRNAs show particulate signals that overlap with Me31B signals (C,F). In contrast, vas mRNA signal does not appear to colocalize with Me31B-containing particles (I). Scale bars, 50 µm and 5 µm (inset).

View larger version (54K): [in a new window]   Fig. 3. Me31B forms a complex with Exu and Yps, and is concentrated in the sponge body. (A-C) A stage 6 egg chamber expressing GFP-Exu (green) was stained for Me31B (red). Me31B colocalizes with Exu. (D-F) A stage 6 egg chamber expressing GFP-Me31B (green) was stained for Exu (red). Exu is colocalized with GFP-Me31B. (G-I) Stage 4 and stage 5 egg chambers expressing GFP-Me31B were stained for Yps (red). Yps is colocalized with GFP-Me31B. Note that Yps is expressed extensively in follicle cells that surround germline cells. Hence, the antibody failed to permeate well into the interior of germline cells. (J) An immunoelectron-microgram of a section for Me31B. The section shown is the cytoplasm of a nurse cell in a stage 9 egg chamber. Me31B signals are enriched in a distinctive cytoplasmic region, which is not surrounded by membrane. We detected 390 signals for Me31B in 356 µm2 areas in sections of nurse cell cytoplasm. Among these, 331 signals (84.9%) were in these distinctive regions, which occupied only 57 µm2 (16%) in the sections. m, mitochondrion. (K) Interaction between Me31B and Exu-Yps complex. Me31B and Yps were immunoprecipitated from ovary extracts in the absence (left) or presence (right) of RNase. Immunoprecipitates were separated by SDS-PAGE, transferred to membrane, and probed with antibodies against Me31B, Yps and Exu. In the presence of RNase A, Me31B-Yps and Me31B-Exu coimmunoprecipitation is diminished (right). Scale bar: 50 µm in A-I; 200 nm in J.

View larger version (14K): [in a new window]   Fig. 4. Genomic organization of me31B locus. A P element insertion, k06607, is located 100 bp upstream of me31B. By mobilizing the P element, three partial deletions within me31B locus (me31B) were isolated. Lethality associated with me31B chromosomes was rescued by the two transgenes (pme9k and PmeAfl) but not by Pme5k. A, AflII; B, BamHI; E, EcoRI; H, HindIII; P, PstI; S, SalI.

View larger version (66K): [in a new window]   Fig. 5. Me31B is essential for oogenesis. Wild-type (A-C) and me31B- (D-I) ovaries were stained with Hoechst (A,D,G) and phalloidin (B,E,H). (D-F) A typical me31B- egg chamber, in which cell membranes of germline cells were collapsed. Ring canals and nurse cell nuclei in these egg chambers form aggregates. (G-H) A stage 10 me31B- egg chamber showing nurse cell nuclei invading the region normally occupied by the oocyte.

View larger version (40K): [in a new window]   Fig. 6. Me31B is dispensable for the transport of Exu, and osk and BicD mRNAs to the oocyte. Arrowheads indicate me31B- egg chambers, which were identified by loss of Vas-GFP (A-D) or Me31B (E-L) signals. In early egg chambers, Exu (B,D), and osk (F,H) and BicD (J,L) mRNAs are all transported to the oocyte even in the absence of Me31B (arrowheads). Particulate signals in nurse cell cytoplasm were detectable in these me31B- egg chambers. Scale bar: 50 µm.

View larger version (68K): [in a new window]   Fig. 7. Osk and BicD signals in nurse cell cytoplasm are increased in me31B- egg chambers. Arrowheads point to me31B- egg chambers, which are identified by loss of Vas-GFP signal (green in A,D,G,J). In these me31B- egg chambers, Osk (red in B,C,E,F) and BicD proteins (red in H,I,K,L) in nurse cell cytoplasm are both detected at higher levels than in me31B+ egg chambers on the same optical sections. Scale bar: 50 µm.

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