Fig 2: A new synthesis for the nucleocytoplasmic distribution mechanism of DDX6.The diagram summarized the two proposed mechanisms governing the nucleocytoplasmic distribution mechanism of DDX6 in this study. (a) The “hitchhiking” model refers to that DDX6 can enter cell nuclei as a passenger on the mitotic chromosomes. (b) The “piggyback” model refers to that 4E-T is sufficient to transport DDX6 via the importin-α/β and the CRM1 pathways in a piggyback manner.

Fig 3: DDX6 interacts with ADARs in the nuclei of human cells. (A) Six regions from the silver-stained gel of an anti-DDX6 IP sample were subjected for MS analysis. Regions containing extra protein bands from the anti-DDX6 IP product are marked in alphabetical order according to their molecular weight (A: >180 kDa; B: 130–180 kDa; C: 120–130 kDa; D: 72–95 kDa; E: 42–50 kDa; F: 35–40 kDa). (B) A total of 36 proteins were identified in a co-IP/MS analysis from samples in regions with various molecular weights. ADAR1 was presented in red. (C,D) Anti-DDX6 immunoprecipitation was conducted in HEK293T cells that overexpressed ADAR1 (C) or ADAR2 (D), treated with +/− RNase A, and was followed by WB analysis using ADAR1, ADAR2, and DDX6 antibodies. Cytoplasmic and nuclear extracts from HEK293T cells were prepared with the Gagnon–Li method. Ten percent of the total cell lysate was used as the input control. TCE, total cell lysates; Nu, nuclear fraction; Cyto, cytoplasmic fraction; IgG, immunoglobin G; 4E-T, positive control for DDX6-IP; GAPDH, cytoplasmic marker; Lamin a/c, nuclear marker.

Fig 4: The putative NES is masked by DDX6 NTD folding.(a) Illustration of the putative L-rich NES in DDX6. The putative NES spans position 151–160 in DDX6 NTD (red segment). The white box represents DDX6 sequence. Sequence alignment of human, mouse and Xenopus DDX6 homologs is shown. Leucine residues matching the consensus L-rich NES pattern is colored in red. Asterisks denote the leucine residues targeted for mutagenesis (L158 and L160; L to A substitution). Hsa: Homo sapiens; Mmu: Mus musculus; Xla: Xenopus laevis. (b) The putative NES is buried in the core of stably folded DDX6 NTD. DDX6 in ATPase-incompatible conformation (4CT5) is visualized, colored, rotated to highlight the structural features of the putative NES. Cylinders represent α-helices; flat arrows represent β-sheets. NES peptide backbone is colored in red; carbon atoms of leucine residues are colored in green. Insets display magnified views of the boxed areas near the putative NES. (c) Illustration depicts the constructs of YFP-DDX6 1–166, YFP-DDX6 NTD, YFP-DDX6 FL, and YFP-DDX6 141–483. Various segments of DDX6 were N-terminally tagged with EYFP. Region of the putative NES is shown in red. All NES mutants (mNES) bear L158A/L160A substitutions. (d) Nucleocytoplasmic distribution of YFP-DDX6 NTD and YFP-DDX6 FL are insensitive to LMB treatment. HeLa cells expressing YFP-4E-T, YFP-DDX6 1–166 WT, YFP-DDX6 NTD WT, YFP-DDX6 FL WT and YFP-DDX6 141–483 WT were treated with 5 ng/mL LMB for 5 hr prior to imaging. Same volume of 70% methanol (MeOH; solvent) was used as control treatment. (e) Nucleocytoplasmic distributions of YFP-DDX6 NTD, YFP-DDX6 FL, and YFP-DDX6 141–483 are insensitive to putative NES mutation. YFP-DDX6 1–166, YFP-DDX6 NTD, YFP-DDX6 FL, and YFP-DDX6 141–483 WT and mNES were transiently expressed in HeLa cells. YFP signal is shown in green. Nuclei were stained with Hoechst (shown in blue). Scale bar = 20 μm. (f) Diagram shows our conclusion for the accessibility of the putative NES.

Fig 5: EIF4ENIF1 and EIF4E protein expression in the ovaries of Eif4enif1WT/flx compared to Eif4enif1WT/Δ mice on day 5. EIF4ENIF1 (green), EIF4E (red) protein expression, and merged in primordial and primary follicles from day 5 Eif4enif1WT/flx (A-F) and Eif4enif1WT/Δ (H-M) ovaries (white arrows show localization). EIF4ENIF1 protein expression is less intense and EIF4E more intense in Eif4enif1WT/Δ compared to Eif4enif1WT/flx ovaries. The red scale bar indicates 100 µm (A–C and H–J) or 50 µm (D–F and K–M).