Krishnakumar P et al. (2018), Functional equivalence of germ plasm organizers.

ECB-ART-48428

PLoS Genet 2018 Nov 06;1411:e1007696. doi: 10.1371/journal.pgen.1007696.

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Functional equivalence of germ plasm organizers.

Krishnakumar P , Riemer S , Perera R , Lingner T , Goloborodko A , Khalifa H , Bontems F , Kaufholz F , El-Brolosy MA , Dosch R .

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The proteins Oskar (Osk) in Drosophila and Bucky ball (Buc) in zebrafish act as germ plasm organizers. Both proteins recapitulate germ plasm activities but seem to be unique to their animal groups. Here, we discover that Osk and Buc show similar activities during germ cell specification. Drosophila Osk induces additional PGCs in zebrafish. Surprisingly, Osk and Buc do not show homologous protein motifs that would explain their related function. Nonetheless, we detect that both proteins contain stretches of intrinsically disordered regions (IDRs), which seem to be involved in protein aggregation. IDRs are known to rapidly change their sequence during evolution, which might obscure biochemical interaction motifs. Indeed, we show that Buc binds to the known Oskar interactors Vasa protein and nanos mRNA indicating conserved biochemical activities. These data provide a molecular framework for two proteins with unrelated sequence but with equivalent function to assemble a conserved core-complex nucleating germ plasm.

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Genes referenced: ddx4 LOC115919910 LOC590297

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	Fig 1. Drosophila Oskar specifies germ cell formation in zebrafish.(A) Scheme of germ cell induction assay. Left panel: Animal view of a 16-cell embryo injected with PGC-reporter into a middle blastomere (blue arrowhead) containing endogenous germ plasm (red dots) or into a somatic cell (corner blastomere; green arrowhead). Right panel: Oblique, dorsal view of a 15-somite stage embryo (18 hours post fertilization, hpf), anterior to the left. Fluorescent germ cells (white arrowhead) emerge by targeting the reporter to a PGC or transforming a somatic cell into a PGC. (B, C) Live 18 hpf embryo after injection of PGC-reporter into a middle (B; 83±2.4%; n = 70) or corner blastomere (C; 31±4.5%; n = 70; p = 0.005). As previously shown, the gfp-nos3’UTR reporter also frequently labeled the midline at this stage [33]. (D, E) Wild-type Buc (aa1-639) reprograms the somatic corner blastomere to the germline (D; wt = 78±2.6%; n = 71), but not mutant Buc (Bucp43). Bucp43 mRNA sequence is identical to wt, but carries a point mutation (Y362STOP) [17] (E; mut = 35±1.6%; n = 68; p = 0.001). (F, G) Xenopus Velo1 acts as a functional homolog (F; 61±3.5%; n = 41) but not zebrafish Ziwi (G). (H, I) Drosophila sOsk induces germ cell formation (H; wt = 78±1.1%; n = 81), but not mutant sOsk084 (aa139-254) (I; mut = 25±8.7%; n = 62; p = 0.01). Scale bar (B-I): 200 μm. (J) Quantification of injection results (three independent experiments for each RNA). Error bars represent standard deviation of the mean. Student’s t-test; P-value: **<0.01.
	Fig 2. Buc and Osk induced PGCs express Vasa protein.Lateral view, anterior to the left of area indicated in icon of 18-somite stage embryo after 16-cell assay with wt Buc(1–639) (A-C), mutant Buc(1–361) (D-F) or sOsk (G-I). Embryos were analyzed for GFP (green) and Vasa (red) protein expression. Arrowheads indicate endogenous PGCs (Vasa positive and GFP negative). Scale bar: 20 μm.
	Fig 3. Drosophila Osk and zebrafish Buc display unrelated protein sequences.(A) Strategy for Osk-Buc sequence comparison using first global and local alignment algorithms, then hidden Markov models (HMM) of both proteins to detect remote homologies, and eventually a comparison of HMM to each other. (B) Graph comparing sequence similarity of Buc with short (aa139-606) and long Osk (aa1-606) isoforms based on global pairwise sequence alignments. Alignment of Buc and Drosophila Vasa (Dm Vasa) serves as negative, whereas zebrafish (Zf Vasa) and Dm Vasa as positive control. (C) Scheme of conserved domains in Buc identified by alignments of 14 protein sequences. Conserved motifs in Osk protein. Red line indicates alternative translation initiation of short Osk at Met139. (D) Scheme indicating significant remote sequence similarity based on HMMER analysis for Buc with zebrafish Dazl and for Osk with the Lotus domain of zebrafish Tdrd7. Numbers indicate amino acid positions.
	Fig 4. Buc and Osk contain intrinsically disordered regions.Predicted protein disorder in (A) Buc, (B) Osk, (C), Vasa, and (D) Ziwi. Disorder disposition (y-axis) plotted against the amino acid residue index (x-axis). Values above the 0.5 threshold (grey bar) show the propensity for disordered regions (bold line). The red line at aa 139 in Osk indicates the alternative translation initiation site for short Oskar. Protein aggregates upon transfection of HEK cells with monomeric GFP (mGFP) fused to (E) Buc, (F) sOsk or (G) unfused. The profiles below the pictures show levels of fluorescent intensity along the line indicated by white dashes. Buc-mGFP (green; H) and Osk-Cherry aggregates (red, I) overlap (J, yellow, white arrowhead). Scale bar (E-J): 10μm.
	Fig 5. Pharmacological disruption of IDR-interactions leads to partially fragmented Buc-GFP aggregates.(A-C) Balbiani body of living Buc-GFP transgenic oocytes, either before (A), after a 30 min treatment with 5% 1,6-hexanediol (HD) (B), or 30 min after washout of the drug (lateral view, animal to the top). Arrowheads in B and B' indicate Buc-GFP granule outside the Balbiani body. Scale bar (A-C): 20 μm (A'-C'): 2 μm. (D-K) Germ plasm of transgenic Buc-GFP embryos after hexanediol treatment (HD). (D, E) lateral view of living 2-cell embryo as shown in boxed area of icon. Control embryos show unfragmented Buc-GFP aggregates (green) (D arrowhead), whereas 5% hexanediol for 30 min leads to fragmentation(arrowheads). (F) Quantification of embryos with unfragmented Buc-GFP in control (Co; 100±0%; n = 20) and embryos treated for 30 min with hexanediol (HD; 35.0±0.8%; n = 20; p = 0.0065). Student's t-test; P-value: <0.01. (G, H) lateral view of living 4-cell embryos. Control embryo with unfragmented BucGFP (green, arrowhead), whereas Buc-GFP stays fragmented 30 min after washout of hexanediol (green; arrowheads). Scale bar (D-H): 100 μm. (I-K) Buc-GFP aggregates in 3 hpf embryos transgenic for Buc-eGFP. (I) The morphology of control (Co) and hexanediol-treated embryos (HD). Lateral view, animal to the top. (I', I'') Fragmented Buc-GFP aggregates (white arrowheads) persist until 3 hpf (I') lateral view, (I'') animal view. (J) Treatment with hexanetriol (HT) also leads to fragmented germ plasm (right embryo in J; animal view). Scale bar (I-J): 500 μm. (K) Quantification of germ plasm fragmentation (more than four puncta) at 3 hpf in controls (Co; 2.2±3.9%; n = 45), hexanetriol (HT; 26.3±11.5; n = 45) and hexanediol (HD; 75.5±3.9; n = 45; p = 1.9e-08). Error bars represent standard deviation of the mean. Student’s t-test; P-value: <0.001. (L-N) Protein aggregates upon transfection of HEK cells with (L) Buc(aa1-601)-GFP (50.32±2.95%; n = 70 percentage of transfected cells showing aggregated GFP signal), (M) Buc(aa1-361)-GFP (77.9±8.8%; n = 89) and (N) GFP (0%; n = 81). Scale bar (L-N): 10μm. (O-Q) Buc aggregation in zebrafish embryos. Embryos at 3 hpf after injection of mRNA encoding wt Buc(aa1-639)-eGFP (O), Buc(aa1-601)-eGFP (P) or eGFP(Q) at the one cell stage (lateral view, animal to the top). Scale bar (O-Q): 200 μm. Note the aggregation of wt Buc (aa1-639) and Buc (aa1-601) compared to GFP (insets; 25x magnification of stippled box). (R-U) IDRs are not sufficient for germ cell induction. Embryos form germ cells (white arrowheads) after injection with wt buc mRNA (aa 1–639) (R; 76.6±2.3%; n = 60), but less with mutant Buc (K; aa1-601) containing most IDRs (S; 35.9±2.6%; n = 60; p = 0.04) or an unrelated IDP (human FUS; T; 0±0; n = 26). Scale bar (J-L): 200 μm. (I) Quantification of injection results (three independent experiments for each RNA). Error bars represent standard deviation of the mean. Student’s t-test; P-value: <0.05.
	Fig 6. Buc binds zebrafish Vasa.(A) Zebrafish homologs of known Oskar binding proteins in the Buc-interactome detected Vasa (Ddx4) and Valois (Wdr77/Mep50), but not Smaug (Samd4b). Enrichment indicates the ratio of unique peptide counts after Buc-GFP pulldown to GFP-control samples. (B) Buc binds to Vasa in vivo during germ cell specification. Immunoprecipitations from 3 hpf H2A-GFP (42 kD) or Buc-GFP (130 kD) transgenic embryos blotted with GFP (green) and Vasa (magenta) (input = 20% of pulldown). (C-F) Buc and Vasa interact in bimolecular fluorescent complementation assays (BiFC). (C-E) live embryos at 3 hpf as indicated by the cartoon on the left, are not fluorescent (green) upon injection of mRNA encoding VC with Vasa-VN (C; 0±0%; n = 67) or Buc-VC with VN (D; 0±0%; n = 56), but form fluorescent Venus protein with Buc-VC and Vasa-VN (E; 86.5±7.5%; n = 53). Scale bar (C-E): 100 μm. (F) Quantification of BiFC assay (three independent experiments for each RNA). Error bars represent standard deviation of the mean. (G-L) Immunostaining of 16-cell stage (G-I) or 3 hpf (J-L) embryo as indicated by the cartoon on the left showing expression of Buc (green) and Vasa (magenta), inset in (L) shows a 10x magnification of the boxed area. Scale bar (G-L): 200μm.
	Fig 7. Zebrafish Vasa induces germ cells and binds to Drosophila Osk.(A-C) 16-cell assay showing germ cell formation (white arrowhead) after injection with vasa mRNA (A; 73.9±5.3%; n = 60; p = 0.01) but not with hermes (B; 22.9±4.8%; n = 60). Scale bar (A, B 200 μm (C) Quantification of injection results (three independent experiments for each RNA). Error bars represent standard deviation of the mean. Student’s t-test; P-value: **<0.01. (D) Western blot of Buc-GFP, Osk-GFP, and GFP-control together with Vasa after in vitro translation (input = 40% of pulldown) and after GFP-pulldown. Vasa (upper panel, magenta in merged panel) interacts with Buc and Osk, but not GFP controls (middle panel, green in merged panel). (M: molecular weight marker lane) (E) Western blot of Buc-GFP, Bucp43-GFP (1–361), and Bucp106-GFP (1–601) (middle panel, green in merged panel) together with Vasa (upper panel, magenta in merged panel) after in vitro translation (input = 40% of pulldown) and after GFP-pulldown. Vasa interacts with Buc, Buc(1–361) and Buc(1–601).
	Fig 8. Buc interacts with RNA.(A) RT-PCR of zebrafish nanos3, SV40, human EF1α and human 18S rRNA after GFP-pulldown (IP) of HEK293 cells either untransfected (-) or transfected with GFP, Osk-GFP or Buc-GFP together with Cherry-nos-3'UTR and Cherry-SV40-3'UTR. RNA levels before GFP-pulldown (Input) show endogenous 18S rRNA and EF1α mRNA, and transfected Cherry-nos-3'UTR and Cherry-SV40-3'UTR. nos3-3'UTR is detected in Osk and Buc samples after GFP-pulldown (IP).–RT: RNA without reverse transcriptase control. (B) Buc(1–361)-GFP does not pulldown nos3-3'UTR. Cherry-nos-3’UTR (+), alone or cotransfected with Osk-GFP, Buc-GFP or Buc(1–361)-GFP including SV40-3'-UTR. (C) RNase treatment (+) prior to GFP pulldown of in vitro translated protein does not disrupt Buc-Vasa interaction. (D) Model for germ plasm formation. Single Buc or Osk molecules (red) aggregate through weak interactions of their intrinsically disordered regions (hooks and loops), until a threshold concentration is reached. This leads to a liquid-liquid phase separation (red haze) to form hydrogel-like germ plasm. The aggregate then recruits Vasa protein (blue) and nanos mRNA (green).

References [+] :

Aguero, Mechanisms of Vertebrate Germ Cell Determination. 2017, Pubmed