Ubiquitin Editing of a Spliceosome Auxiliary Factor By TRAF6 Links Chronic TLR Signaling with Hematopoietic Defects and Myelodysplasia
Authors: Fang Jing , Bolanos Lyndsey , Choi Kwang-Min , Liu Xiaona , Christie Susanne , Akunuru Shailaja , Kumar Rupali , Greis Ken , Stoilov Peter , Filippi Marie-Dominique , Maciejewski Jaroslaw P. , Garcia-Manero Guillermo , Weirauch Matthew , Salomonis Nathan , Geiger Hartmut , Zheng Yi , Starczynowski Daniel 
Published: October 2019

Abstract
Toll-like receptors (TLR) are known for regulating myeloid homeostasis and response to infection, but chronic activation of TLR pathways can also lead to hematopoietic stem and progenitor cell (HSPC) dysfunction. Furthermore, mutations that lead to constitutive activation of TLR pathways contribute to premalignant hematologic conditions, such as myelodysplastic syndromes (MDS); however, the underlying cellular and molecular mechanisms are unknown. As a means of chronically activating TLR signaling within HSPCs, we generated a mouse model by elevating expression of TRAF6 in hematopoietic cells (Vav-TRAF6). TRAF6 is a downstream TLR-effector with ubiquitin (Ub) E3 ligase activity, and is overexpressed in MDS HSPCs. Vav-TRAF6 mice developed progressive leukopenia and anemia, and exhibited myeloid skewing and dysplasia. Eventually, over half of Vav-TRAF6 mice succumbed to a bone marrow (BM) failure associated with MDS. Despite increased frequencies of immunophenotypic HSPCs in the BM, Vav-TRAF6 HSPCs are functionally defective as evidenced by impaired colony formation and reduced in vivo competitiveness. The hematopoietic phenotype due to TRAF6 overexpression was still manifest upon transplantation, indicating that the effect is hematopoietic cell intrinsic. Consistent with the cellular effects observed with chronic TLR activation, elevated TRAF6 expression results in MDS/BMF by altering intrinsic HSPC properties.
Gene expression and exon level analyses revealed that Vav-TRAF6 HSPCs exhibit discrete and durable alterations in RNA splicing patterns. The family of small G-protein GTPases emerged as a relevant pathway whose activity is affected by missplicing of Arhgap1, a GTPase activating protein. Aberrant skipping of Arhgap1 exon 2 resulted in reduced Arhgap1 protein and constitutive Cdc42 GTPase activation. Inhibition of Cdc42 activity with a pharmacological inhibitor partially reversed myeloid-biased differentiation of Vav-TRAF6 HSPCs in vivo, indicating that missplicing of Arhgap1 and increased Cdc42 activity accounts for several HSPC defects.
To identify the mechanism underlying TRAF6-induced RNA splicing, we employed a global Ub-enrichment screen for novel TRAF6 substrates, and uncovered hnRNPA1, an RNA-binding protein that regulates exon usage. hnRNPA1 is ubiquitinated by TRAF6 adjacent to and within its first RNA-binding domain. hnRNPA1 binding sites were significantly enriched within misspliced exons in Vav-TRAF6 HSPCs and in primary human AML samples with elevated TRAF6 expression, indicating that TRAF6 overexpression induces exon skipping via hnRNPA1. The requirement of hnRNPA1 in TRAF6-induced exon skipping was confirmed as knockdown of hnRNPA1 significantly reduced Arhgap1 exon 2 skipping in Vav-TRAF6 HSPC. Moreover, depletion of hnRNPA1 reversed Vav-TRAF6 hematopoietic defects in vivo, unequivocally validating the importance of hnRNPA1 in TRAF6-mediated exon skipping and function of HSPCs.
Our findings uncover a novel mechanism by which sustained TLR signaling, via TRAF6-mediated ubiquitination of hnRNPA1, alters RNA splicing and contributes to MDS-associated HSPC defects in part by activating Cdc42. These results indicate a novel function for Ub signaling in coordinating transcriptional initiation and alternative splicing by TLR signaling pathway within the immune system and in premalignant hematologic diseases, such as MDS.

Disclosures
Starczynowski: Celgene Corporation: Research Funding.