Aguero T , Zhou Y , Kloc M , Chang P , Houliston E , King ML .

R01 GM102397 NIGMS NIH HHS , P30 CA016672 NCI NIH HHS

	Figure 1. Hermes/Rbpms protein and nanos RNA localize within germinal granules unique to germ plasm. Stage I oocyte. (A) Immunofluorescence (IF) and confocal microscopy showing Hermes/Rbpms protein (green) is ubiquitous in stage I oocyte including the mitochondrial cloud (MC), nucleus (N) and ooplasm; microtubules (red). Enlarged image from (A) showing microtubule organization around MC (B) and Hermes/Rbpms (C); Note how Hermes/Rbpms is enriched within the germ plasm region of the MC; (D) Electron micrograph showing a region of germ plasm within the MC of stage I oocyte. Red arrows indicate two germinal granules; (E) Nanos RNA is localized within germinal granules in stage I oocytes as is Hermes/Rbpms protein (F). Electron microscopy in situ hybridization with antisense Digoxigenin labeled nanos RNA was visualized with nanogold conjugated anti-Dig antibody and silver enhancement. Electron microscopy immunostaining with Hermes/Rbpms polyclonal antibody [49], anti-rabbit nanogold conjugated secondary antibody and silver enhancement. Scale bars are as indicated. GG (germinal granules); M (mitochondria); N (nucleus).
	igure 2. Hermes/Rbpms specifically associates with nanos RNA in a UGCAC dependent manner. (A) Hermes/Rbpms interacts with nanos but not Vg1 RNA. Vg1 3′UTR Localization Experimental design is shown at top. Vg1 Localization Signal (LS), nanos1 3′UTR, nanos Mitochondrial Cloud Localization Signal (MCLS), and a nanos mutant MCLS (mut) were tested for Hermes/Rbpms binding. Individual RNAs were immobilized on AADA agarose beads and mixed with in vitro translated Hermes/Rbpms protein labeled with 35S-methionine. Bound proteins were analyzed by SDS-PAGE and autoradiography. Galactosidase (Gal) RNA served as a negative control. Hermes/Rbpms only bound the nanos1-3′UTR and only when reactions included stage I/II oocyte extracts and not reticulocyte lysates alone. Note: the 3′UTR binds more Hermes/Rbpms than the MCLS alone suggesting the GGLE may also bind Hermes/Rbpms; (B) Both the MCLS and GGLE are required for wild-type levels of Hermes/Rbpms binding and require stage I oocyte extract. The deletion mutants diagramed were immobilized on AADA beads and analyzed as described in Methods. Error bars indicate standard deviation of three experiments. Hermes/Rbpms binding to the full-length nanos 3′UTR is set at 100%. Note: both regions contribute to Hermes binding.
	Figure 3. The carboxyl terminal 34 amino acids of Hermes/Rbpms are required for nanos1 binding and for Hermes/Rbpms dimerization. (A) Three deletion mutants of Hermes/Rbpms are diagramed showing the RRM domain and the hydrophilic carboxyl terminal 34 amino acids (red box). 35S-methionine labeled Hermes/Rbpms and its deletion mutants were analyzed for their ability to be pulled-down by nanos1 3′UTR immobilized on AADA beads. Wild-type and mutant protein (input) used in reaction and bound are shown by SDS-PAGE. Note that only the last 34 amino acids were required for nanos binding in the presence of the Hermes/Rbpms RRM; (B) The presence of Hermes/Rbpms homodimers was detected by autoradiography as two bands. Note that Hermes/Rbpms missing the terminal 34 amino acids failed to form a dimer. As nanos RNA was not present, Hermes/Rbpms formed homodimers in the absence of RNA; (C) C-terminal region involved in Hermes/Rbpms homodimerization and binding to the nanos 3'UTR. Alignment of conserved terminal 34 amino acids (AA) of Human and Xenopus Hermes/Rbpms proteins. An * (asterisk) indicates a fully conserved AA. Black dots indicate conservation between groups of strongly similar properties (scoring > 0.5 in the Gonnet PAM 250 matrix). A white dot indicates weakly similar properties (scoring ≤ 0.5 in the Gonnet PAM 250 matrix), Clustal Omega, EMBL-EBI, UK. Table shows the high level of identity and similarity between frog and human Hermes/Rbpms proteins.
	Figure 4. Hermes/Rbpms interacts with another Nanos1 binding protein: To identify proteins that might interact with Hermes/Rbpms during early oogenesis, a Xenopus cDNA library (Clontech) was screened using a yeast two-hybrid (Y2H) system. (A) Full length Hermes/Rbpms fused with the DNA binding domain (BD) of the transcription factor Gal4 served as bait. Candidate interacting proteins were fused to the Gal4-activation domain (AD) and served as prey. The reporter gene B-galactosidase, driven by the Gal4 binding site, was positively transcribed as the result of Hermes/Rbpms and hnRNP I (heterogeneous nuclear ribonucleoprotein I) interaction. Two proteins involved in yeast glucose metabolism, Sfn4 and Sfn1, were used as controls to discard false positive results. Snf4/Gal4-BD was used as a bait and Snf1/Gal4-AD as a prey to check false interactions with hnRNP I and Hermes/Rbpms protein respectively; (B) The C-terminal 34 amino acids (in red) required for Hermes/Rbpms to dimerize and bind nanos RNA are not required to interact with hnRNP I. Hermes/Rbpms protein lacking the terminal 34 AA was used as bait and hnRNP I as prey. Induction of B-galactosidase indicated a positive interaction between bait and prey.
	Figure 5. Cellular distribution of Hermes/Rbpms, hnRNP I, Vg1RBP/Vera, and Xpat proteins in the stage I/II oocytes. Confocal images showing immunofluorescence (IF) of stage I/II oocyte with (A) anti-Hermes/Rbpms (green) and (A’) anti-Xpat (red) antibodies; (A’’) Superimposed image. White arrows indicate Xpat and Hermes/Rbpms particles are not identical; (B) Superimposed images of hnRNP I (green) and GRP94 (red) proteins. Co-staining with anti-GRP94 reveals the ER enriched within the MC, cortex, and perinuclear region. Note that hnRNP I is excluded from the MC but is present in the nucleus and cytoplasm; (C) Merged images of endogenous Vg1RBP/Vera (red); or (D) hnRNP I (red) with Alexa 488-labeled nanos 3′UTR injected 20 h before fixation (green). Note that while nanos RNA localizes to the MC, both Vg1RB/Vera and hnRNP I are excluded from it. N: nucleus; MC: mitochondrial cloud. Scale bars are as indicated.
	Figure 6. Hermes/Rbpms associates with nanos but not Vg1 RNA in the nucleus. (A) Overall experimental design. Myc-Hermes/Rbpms mRNA was injected into stage I/II oocytes. After culturing for two days, either myc-Hermes/Rbpms protein was immunoprecipitated (IP) with anti-myc antibody from whole oocytes only or the nuclear (N) and cytoplasmic (C) fractions were manually isolated before IP with anti-myc antibody. In either design, RNA was extracted from the resulting pellet and analyzed with specific primers for Vg1 or nanos RNA by RT-PCR; (B) Myc-Hermes/Rbpms interacts with nanos but not Vg1 RNA in vivo. Control RT-PCR with whole oocytes shows that nanos and Vg1 RNAs were present. Control western blot shows that myc-Hermes/Rbpms was translated after injection; (C) The nuclear and cytoplasmic fractions were manually isolated from stage I/II oocytes. Tubulin (cytoplasmic marker) and nucleoplasmin (nuclear marker) were detected by western blot analysis and show clean separation; (D) Late and Early Pathway RNAs accumulate in nucleus concurrently. RNAs were analyzed by RT-PCR using specific primers; (E) Western blot analysis showed newly translated Hermes/Rbpms in the nucleus; (F) Hermes/Rbpms interacts with nanos RNA in the nucleus, but not Vg1 or VegT. WO: whole oocyte; Uninjected oocytes served as negative controls. Blue sphere (MC); N (nucleus).
	Figure 2. Hermes/Rbpms specifically associates with nanos RNA in a UGCAC dependent manner. (A) Hermes/Rbpms interacts with nanos but not Vg1 RNA. Vg1 3′UTR Localization Experimental design is shown at top. Vg1 Localization Signal (LS), nanos1 3′UTR, nanos Mitochondrial Cloud Localization Signal (MCLS), and a nanos mutant MCLS (mut) were tested for Hermes/Rbpms binding. Individual RNAs were immobilized on AADA agarose beads and mixed with in vitro translated Hermes/Rbpms protein labeled with 35S-methionine. Bound proteins were analyzed by SDS-PAGE and autoradiography. Galactosidase (Gal) RNA served as a negative control. Hermes/Rbpms only bound the nanos1–3′UTR and only when reactions included stage I/II oocyte extracts and not reticulocyte lysates alone. Note: the 3′UTR binds more Hermes/Rbpms than the MCLS alone suggesting the GGLE may also bind Hermes/Rbpms; (B) Both the MCLS and GGLE are required for wild-type levels of Hermes/Rbpms binding and require stage I oocyte extract. The deletion mutants diagramed were immobilized on AADA beads and analyzed as described in Methods. Error bars indicate standard deviation of three experiments. Hermes/Rbpms binding to the full-length nanos 3′UTR is set at 100%. Note: both regions contribute to Hermes binding.

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