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Ubiquitination of hnRNPA1 by TRAF6 links chronic innate immune signaling with myelodysplasia

Nature Immunology volume 18, pages 236–245 (2017)Cite this article

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A Corrigendum to this article was published on 22 March 2017

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Abstract

Toll-like receptor (TLR) activation contributes to premalignant hematologic conditions, such as myelodysplastic syndromes (MDS). TRAF6, a TLR effector with ubiquitin (Ub) ligase activity, is overexpressed in MDS hematopoietic stem/progenitor cells (HSPCs). We found that TRAF6 overexpression in mouse HSPC results in impaired hematopoiesis and bone marrow failure. Using a global Ub screen, we identified hnRNPA1, an RNA-binding protein and auxiliary splicing factor, as a substrate of TRAF6. TRAF6 ubiquitination of hnRNPA1 regulated alternative splicing of Arhgap1, which resulted in activation of the GTP-binding Rho family protein Cdc42 and accounted for hematopoietic defects in TRAF6-expressing HSPCs. These results implicate Ub signaling in coordinating RNA processing by TLR pathways during an immune response and in premalignant hematologic diseases, such as MDS.

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Figure 1: Overexpression of TRAF6 results in HSPC defects.
Figure 2: TRAF6 regulates ubiquitination of RNA-binding proteins and RNA splicing.
Figure 3: TRAF6 ubiquitinates hnRNPA1.
Figure 4: hnRNPA1 contributes to the hematopoietic phenotype in Vav-TRAF6 mice.
Figure 5: TRAF6 regulates RNA splicing of Arhgap1.
Figure 6: Exclusion of exon 2 results in reduced Arhgap1 protein and Cdc42 activation.
Figure 7: Cdc42 contributes to HSPC defects in Vav-TRAF6 mice.
Figure 8: Cdc42 activation is associated with primary human MDS.

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  • 23 January 2017

    In the version of this article initially published online, the 16th author's surname was spelled incorrectly as 'Salamonis'. The correct spelling is 'Salomonis'. The error has been corrected in the PDF and HTML versions of this article.

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Acknowledgements

We thank members of the Starczynowski laboratory for discussions. We thank J. Bailey and V. Summey for assistance with transplantations (Comprehensive Mouse and Cancer Core). We also thank L. Salati (West Virginia University) for advice on the RIP assays, and H. Singh (CCHMC) for helpful discussion. MDSL cells were kindly provided by K. Tohyama (Kawasaki Medical University). Traf6-floxed mice were kindly provided by Y. Choi (University of Pennsylvania). This work was supported by Cincinnati Children's Hospital Research Foundation, American Society of Hematology (ASH), National Institute of Health (RO1HL111103, RO1DK102759, RO1HL114582), Gabrielle's Angel Foundation for Cancer Research, and Edward P. Evans Foundation grants to D.T.S. D.T.S. is a Leukemia Lymphoma Society Scholar. Cores are supported through the NIDDK Centers of Excellence in Experimental Hematology (P30DK090971).

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Author notes

  1. Jing Fang

    Present address: Present address: Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, South Carolina, USA.,

Authors and Affiliations

  1. Division of Experimental Hematology and Cancer Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA

    Jing Fang, Lyndsey C Bolanos, Kwangmin Choi, Xiaona Liu, Susanne Christie, Shailaja Akunuru, Rupali Kumar, Marie-Dominique Filippi, Hartmut Geiger, Yi Zheng & Daniel T Starczynowski

  2. Division of Pathology and Laboratory Medicine, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA

    Dehua Wang

  3. Center for Autoimmune Genomics and Etiology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, USA

    Xiaoting Chen & Matthew T Weirauch

  4. Department of Cancer Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA

    Kenneth D Greis & Daniel T Starczynowski

  5. Department of Biochemistry, West Virginia University, Morgantown, West Virginia, USA

    Peter Stoilov

  6. Department of Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, Ohio, USA

    Jaroslaw P Maciejewski

  7. Department of Leukemia, Maryland Anderson Cancer Center, Houston, Texas

    Guillermo Garcia-Manero

  8. Division of Biomedical Informatics, Cincinnati Children's Hospital Research Medical Center, Cincinnati, Ohio, USA

    Matthew T Weirauch & Nathan Salomonis

  9. Division of Developmental Biology, Cincinnati Children's Hospital Research Medical Center, Cincinnati, Ohio, USA

    Matthew T Weirauch

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Contributions

J.F., L.C.B., M.-D.F., K.D.G., H.G., Y.Z. and D.T.S. designed and performed experiments. X.L., S.C., S.A. and R.K. provided technical assistance. K.C. provided bioinformatics support. X.C., N.S. and M.T.W. performed the RNA splicing and exon binding analysis. P.S. provided reagents and advice on RNA splicing. D.W. provided histopathology advice. K.G. performed the mass spectrometry analysis. G.G.-M. and J.P.M. provided patient samples. J.F. and D.T.S. wrote the manuscript.

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Correspondence to Daniel T Starczynowski.

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Integrated supplementary information

Supplementary Figure 1 Generation of a hematopoietic-specific TRAF6 overexpression mouse to mimic expression of TRAF6 in human MDS.

(a) A schematic of the Vav-TRAF6 construct used for pronuclear injection to generate Vav-TRAF6 transgenic mice. The 5’ and 3’ Vav regulatory elements, simian virus 40 (SV40) intron and polyadenlyation signal are indicated. A FLAG-tagged human TRAF6 cDNA was cloned downstream of a Vav promoter. (b) Immunoblotting for TRAF6 in spleen and liver cells from WT (FVB/NJ) and Vav-TRAF6 mice. (c) Immunoblotting for TRAF6 in Lin- BM cells from WT and 3 independent genetic Vav-TRAF6 founder mice on the FVB/NJ or C57/Bl6 background. (d) Summary of spleen weight from WT (FVB/NJ) and moribund Vav-TRAF6 mice (left). Representative spleens from WT (FVB/NJ) and Vav-TRAF6 mice (right). (e) Summary of CD11b/Gr1 (myeloid), Ter119 (erythroid), B220 (B lymphocytes), and CD3 (T lymphocytes) immunophenotypic proportions within the spleen of WT (FVB/NJ; n = 4) and moribund Vav-TRAF6 mice (n = 4).

Supplementary Figure 2 TRAF6 overexpression results in HSPC defects following competitive BM transplantations.

(a) Experimental overview of competitive BMT. BM MNC or LT-HSC (CD45.2+) from WT (C57Bl/6) or Vav-TRAF6 mice were mixed with competitor BM cells (CD45.1+) at 1:1 ratio, and transplanted into lethally-irradiated recipient mice (BoyJ) as the primary (10) cBMT. BM cells were recovered from recipients at 5 months post 10 cBMT, and transplanted into lethally-irradiated recipient mice (BoyJ) for the secondary (20) cBMT. (b) Peripheral blood chimerism of donor-derived (CD45.2+) MNC was determined after 10 and/or 20 cBMT using MNC or LT-HSC. (n > 5 per group). (c) BM chimerism of myeloid (CD11b+) and lymphoid (CD3+ and B220+) cells of donor-derived peripheral blood (CD45.2+) was examined after the 20 BMT (n > 5 per group). (d) BM chimerism of HSPC was determined after the 10 cBMT. (n > 6 per group). (e) Proportion of myeloid (CD11b+) and lymphoid (CD3+ and B220+) cells in the competitor cell-derived compartment (CD45.1+) after competitive transplantation with either BM mononuclear cells (MNC, left) or LT-HSC (right). (n > 7 mice per genotype). *, P < 0.05.

Supplementary Figure 3 TRAF6 overexpression alters myeloid differentiation, innate immune, and hematopoietic stem/progenitor gene profiles.

(a) Differentially expressed genes in WT (FVB/NJ; n = 4) and Vav-TRAF6 (n = 3) LSK at 6 months of age. (b) Gene set enrichment analysis (GSEA) performed on LSK RNA isolated from WT and Vav-TRAF6 mice.

Supplementary Figure 4 hnRNPA1 is an ubiquitin substrate of TRAF6.

(a) A schematic representation of the comparative Ub (di-Glycine) proteomic assay to identify novel TRAF6 ubiquitinated substrates in TF1 cells with (+) and without (-) TRAF6 (left panel), and in vitro ubiquitin reconstitution assay on individual protein substrates (right panel). (b) TF1 cells expressing a doxycyclin (DOX)-inducible shRNA targeting TRAF6 were used for comparative Ub (di-Glycine) capture proteomics. The table contains the number of identified peptides at each step of the process (upper). The Venn diagram represents the final number of unique peptides in each experimental group (lower). (c) Immunoprecipitated (IP) hnRNPA1 was immunoblotted (IB) for Ub in TF1 cells expressing a DOX-inducible TRAF6 shRNA. (+ DOX, TRAF6 knockdown). (d) HEK293 cells transfected with HA-Ub, MYC-hnRNPA1, and/or FLAG-TRAF6 were IB for IP MYC-hnRNPA1 (anti HA-Ub).

Supplementary Figure 5 TRAF6 affects RNA isoform expression and splicing in HSPC.

(a) FIRMA Index values and RT-PCR analysis (using primers flanking the skipped exon) of the indicated cassette exons in Vav-TRAF6 and WT (FVB/NJ) LSK (left) and LPS-treated WT Lin- BM cells (right). Values under the plot represent the short isoform as a percentage of the short and long isoforms. (b-d) Endogenous Cep164 exon 7 usage as measured by qRT-PCR using primers to the exon 6-8 junction (as depicted in the schematic on the right) and normalized to total Cep164 in WT Lin- BM cells stimulated with 10 ng/mL of LPS (b), LSK and Lin- BM cells from WT (FVB/NJ) and Vav-TRAF6 mice (c), and TRAF6-deficient Lin- BM cells (C57Bl/6 Traf6WT/WT;Mx1Cre and Traf6fl/fl;Mx1Cre mice treated with PolyIC) stimulated with 10 ng/mL of LPS (d). (n > 3 per experimental group). *, P < 0.05.

Supplementary Figure 6 Loss of Arhgap1 contributes to myeloid dysplasia.

(a) Wright-Giemsa-stained peripheral (PB) blood and bone marrow (BM) from WT (C57Bl/6) and Arhgap1-deficient (Arhgap1+/-) mice. Red arrows indicate dysplastic myeloid cells with Pseudo-Pelger Huet anomaly (upper) and neutrophils with hypersegmentation (lower). (b) Percent BM neutrophil dysplasia was determined in age-matched WT (n = 3) and Arhgap1+/- (Gap1+/-) (n = 6) mice. At least 100 cells were examined for each mouse. *, P < 0.05.

Supplementary Figure 7 Role of hnRNPA1 in TRAF6-mediated exon skipping of ARHGAP17 in human MDS.

(a) Schematic representation of the human ARHGAP17 gene structure with the approximate position of exons and introns. Overview of ARHGAP17 exon usage is shown for normal expression (black lines) and in THP1 cells stimulated with LPS (P = 0.0087) or MDSL cells (red lines). Below is the predicted protein products from ARHGAP17-DEx18 with the approximate positions of the BAR, Gap, and SH3 domains. (b) Knockdown of human hnRNPA1 by 2 independent shRNAs (shhnRNPA1-1 [TRCN0000006583] and shhnRNPA1-2 [TRCN0000006586]) was measured by qRT-PCR in HEK293 and MDSL (left) and by immunblotting in HEK293 (right). (c) RT-PCR analysis using primers flanking endogenous human ARHGAP17 exon 18 (as depicted in the schematic on the right) in HEK293 or MDSL cells following knockdown of hnRNPA1 (shA1). (d) Immunoblotting of ARHGAP17 and hnRNPA1 in MDSL cells transduced with independent shRNAs targeting hnRNPA1. (e) Overview of TRAF6 regulation of RNA splicing function during transient and chronic TLR signaling: TRAF6 ubiquitinates hnRNPA1 at the RNA recognition domain. Ubiquitinated hnRNPA1 binds directly and/or indirectly to select exons and results in increased exon exclusion and isoform expression. One of the signaling pathways affected by differential splicing is the small G-protein GTPase family, including Cdc42. Altered splicing of Arhgap1 (a negative regulator of Cdc42) results in active Cdc42, which in part contributes to the HSPC defects and myeloid dysplasia associated with BM failure.

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Fang, J., Bolanos, L., Choi, K. et al. Ubiquitination of hnRNPA1 by TRAF6 links chronic innate immune signaling with myelodysplasia. Nat Immunol 18, 236–245 (2017). https://doi.org/10.1038/ni.3654

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