J Dev Biol
March 1, 2016;
Hermes (Rbpms) is a Critical Component of RNP Complexes that Sequester Germline RNAs during Oogenesis.
The germ cell
lineage in Xenopus is specified by the inheritance of germ plasm
that assembles within the mitochondrial cloud
or Balbiani body in stage I oocytes. Specific RNAs, such as nanos1
, localize to the germ plasm
has the essential germline function of blocking somatic gene expression and thus preventing Primordial Germ Cell
(PGC) loss and sterility. Hermes/Rbpms protein and nanos RNA co-localize within germinal granules, diagnostic electron dense particles found within the germ plasm
. Previous work indicates that nanos accumulates within the germ plasm
through a diffusion/entrapment mechanism. Here we show that Hermes/Rbpms interacts with nanos through sequence specific RNA localization signals found in the nanos-3''UTR. Importantly, Hermes/Rbpms specifically binds nanos, but not Vg1
RNA in the nucleus
of stage I oocytes. In vitro binding data show that Hermes/Rbpms requires additional factors that are present in stage I oocytes in order to bind nanos1
. One such factor may be hnRNP
I, identified in a yeast-2-hybrid screen as directly interacting with Hermes/Rbpms. We suggest that Hermes/Rbpms functions as part of a RNP complex in the nucleus
that facilitates selection of germline RNAs for germ plasm
localization. We propose that Hermes/Rbpms is required for nanos RNA to form within the germinal granules and in this way, participates in the germline specific translational repression and sequestration of nanos RNA.
J Dev Biol
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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 , 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.