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. 2022 Dec 7;42(3):e111364. doi: 10.15252/embj.2022111364

Figure 7. zParn co‐phase separates into zRbm14 condensates to efficiently deadenylate their content maternal mRNAs.

Figure 7

  • A
    Experimental scheme for poly(A) length assays. Two sets of PCR primers (colored arrows) were used to amplify total transcripts and poly(A)‐transcripts, respectively, for a certain gene as illustrated.
  • B, C
    Depletion of zRbm14 resulted in accumulation of poly(A)‐containing maternal mRNAs. Total RNAs purified from the indicated zebrafish embryos were subjected to poly(A) length assays. One set of representative PCR results for org and trip10a (B) and quantification results from three independent experiments (C) are presented. As the PCR products of the poly(A)‐transcripts mainly emerged as a single band, only intensities of this major band were measured. Refer to Fig EV4A and B for additional results.
  • D
    zParn associated with zRbm14b. GFP‐zRbm14b was co‐expressed respectively with the indicated RFP fusion proteins in HEK293T cells. Co‐immunoprecipitations (co‐IP) were performed using anti‐GFP beads. Luciferase (Luc)‐RFP served as a negative control.
  • E, F
    zParn markedly promoted LLPS of zRbm14b by interplaying with zIDR. Purified His‐tagged RFP or zParn‐RFP was mixed with His‐tagged GFP or GFP‐zRbm14b or mutants (E). Parn‐RFP was mixed with decreasing concentrations of GFP‐zRbm14b (F).
  • G, H
    zParn was highly expressed from 4‐ to 32‐cell stages and enriched in zRbm14 condensates. Immunoblotting (G) and immunostaining (H) were performed using a rabbit anti‐zebrafish Parn antibody. Lysates from 5 embryos were loaded in each lane in (G). α‐tubulin served as internal controls. Representative single optical sections are presented in (H). Chromatin DNA was stained with DAPI. Arrows point to typical regions abundant in condensates.
  • I
    Experimental scheme for deadenylation assays. The 5′ and 3′ ends of 10m6A were capped and polyadenylated in vitro to generate a test mRNA, Cap‐10m6A‐An, for the assays.
  • J
    zRbm14b and zParn efficiently co‐phase separated in deadenylation experiments. The addition of SDS disrupted the condensates to terminate the deadenylation reaction.
  • K, L
    Co‐phase separation with zRbm14b markedly enhanced the deadenylation activity of zParn. RNAs extracted from the indicated samples were subjected to immuno‐northern blotting using an antibody against m6A (K). Relatively proportions of Cap‐10m6A‐A0 in total RNAs (L) were quantified from five sets of independent results.
  • M
    A summarizing model. Refer to main text for details.

Data information: Micrographs for in vitro assays (E, F, and J) were taken manually under a wide‐field microscope. Sometimes some condensates moved during imaging, resulting in a positional shift between their GFP and RFP images. Quantification results are presented as mean ± SD. Unpaired two‐sided Student's t‐test against control group: ns, no significance; *P < 0.05; **P < 0.01; ***P < 0.001. Note that the t‐test cannot be performed for the 2.5‐hpf samples in (C) because the relative mRNA levels of control samples were set as 1 and thus have no error bars.

Source data are available online for this figure.