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. 2023 Jan;37(1):235-239.
doi: 10.1038/s41375-022-01753-4. Epub 2022 Nov 8.

Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue

Jeong-Ah Kim  1 , Siqi Shen  2 , Daniel R Matson  1 , Lauren N Lovrien  3 , Kelcy J Smith-Simmer  4 , Sunduz Keles  2 , Jane E Churpek  5 , Emery H Bresnick  6
Affiliations

Discriminating activities of DEAD-Box Helicase 41 from myeloid malignancy-associated germline variants by genetic rescue

Jeong-Ah Kim et al. Leukemia. 2023 Jan.
No abstract available

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Conflict of interest statement

Competing Interests

The authors declare no competing interests.

Figures

Fig. 1:
Fig. 1:. Family histories and structural predictions of myeloid malignancy-associated DDX41 variants.
A K331del and R293H pedigrees. Probands are indicated with an arrow. B Domain structure of human DDX41. Germline and somatic mutations are indicated within the corresponding domains. C Sanger sequencing data of probands in each family. D Predicted structure of mutant human DDX41 by AlphaFold and 3-D images are created using Mol* viewer (https://molstar.org/viewer/). The residues forming the hydrogen bond and surrounding residues are indicated in black. Residues where this interaction is lost due to mutation are indicated in red. E Confidence of predicted structure from AlphaFold analysis.
Fig. 2:
Fig. 2:. Quantitative genetic rescue assay discriminates DDX41 and human myeloid malignancy-associated variant activities.
A Genetic rescue system. Three CRISPR/Cas9 RNP complexes for Ddx41 were electroporated individually into HoxB-immortalized (hi)-progenitors, and clonal lines were isolated. Retrovirus expressing GFP or GFP and DDX41 or a human disease-associated DDX41 variant were infected into hi-Ddx41+/+ and hi-Ddx41+/− cells. GFP-positive cells were isolated by flow cytometry and analyzed by qRT-PCR and RNA-seq. Protein was analyzed by semi-quantitative western blotting. B Exons corresponding to representative domains and target loci of crRNAs on murine Ddx41. C Sanger sequencing of genomic DNA at the edited region of hi-Ddx41+/− clonal lines. The crRNA used to generate each clone is indicated in parenthesis. D DDX41 protein levels in hi-Ddx41+/+ and hi-Ddx41+/− cells were analyzed by semi-quantitative Western blotting using two different antibodies (mouse monoclonal anti-DDX41 antibody (2F4), Novus Biologicals, and our rabbit polyclonal anti-DDX41 antibody). Each clone number indicated corresponds to each clone in Fig. 2C. E Representative Western blots of Flag-tagged DDX41 and variants and endogenous DDX41 using anti-DDX41 antibody in genetic rescue assay. F Quantitative analysis of DDX41 protein of Fig. 2E (n=3 per group). G Subcellular localization of DDX41 and variants by confocal fluorescence microscope using anti-Flag antibody. H mRNAs regulated by DDX41 genetic rescue in hi-Ddx41+/− cells. Heatmap with TPM values of DDX41-regulated mRNAs from total RNA-Seq (n=3 per group). I qRT-PCR analysis of DDX41-regulated mRNAs (n=6 per group). Statistics were calculated using one-way ANOVA, followed by Tukey’s test; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001. J Monocytic and granulocytic populations of live GFP+ cells with CD11b and CD115 surface markers detected by flow cytometry (n=6 per group). Statistics were calculated using one-way ANOVA, followed by Tukey’s test; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001; ****, P ≤ 0.0001.
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References

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