RNA Binding Protein Syncrip Is Required for the Low-Output HSC By Sustaining Proteome Quality

RNA binding proteins (RBPs) have been increasingly recognized as an important class of regulators of normal and malignant hematopoiesis. However, the exact function and underpinning mechanisms of the RBPs that govern hematopoietic stem cells (HSCs) remains poorly characterized. We had previously identified SYNCRIP as a critical RBP that controls leukemia stem cell program in myeloid leukemia. Here, using the novel murine genetic conditional knockout (cKO) model, we delineated the role of SYNCRIP in regulating the low-output HSC. We developed a Syncrip cKO allele and crossed Syncrip^f/f mice to the interferon (IFN) -a-inducible Mx-1-Cre mice to create Syncrip^f/f Mx-1-Cre+. We consistently obtained near complete depletion of SYNCRIP 3 weeks after two consecutive Poly(I:C) injections. We observed that SYNCRIP is dispensable for static hematopoiesis and Syncrip KO animals showed equivalent number and frequencies of stem and progenitor cells (Lin-Sca+cKit+ (LSK)- LT-HSC (CD48-CD150+); MPP1 (CD48-CD150-); MPP2 (CD48+CD150+); MPP4 (CD48-CD150-)). However, KO Syncrip^D/D deficient cells were outcompeted by WT Syncrip^f/f cells in the transplantation setting (bone marrow (BM) chimerism WT (n=9) 38% ± 7.8% vs. KO (n=9) 2.7% ± 0.8%, p<0.001 at 16 weeks post-transplant) and completely lost their ability to repopulate in secondary recipient animals (WT (n=5) 58% ± 7.4% vs. KO (n=5) 7.2 %± 2.9%, p<0.001 at 16 weeks post-transplant). These data strongly indicate that SYNCRIP is critical for maintenance of long-term self-renewal of HSCs. To decipher the effect of Syncrip deletion on the transcriptomic changes in different cell types upon Syncrip loss, we performed single cell RNA sequencing analysis (scRNA-seq) of sorted LK cells (Lin-cKit+ cells) from KO Syncrip^D/D (n=3) vs. WT Syncrip ^f/f (n=3) mice. While there is no significant change in frequencies of stem and progenitor compartments, we found defective trajectory from the HSC that is closely identified as low-output HSC based on previously performed barcoding studies. We observed a strong activation of cellular response to stress and unfolded proteins, in particular the HSF1-dependent pathways upon Syncrip depletion specifically within the HSC population. To further investigate the impacts of SYNCRIP loss in the HSC unfolded protein stress response, we evaluated unfolded proteins in cells using tetraphenylethene maleimide (TMI)-based flow cytometry. The abundance of accessible thiols in unfolded proteins, which is bound by TMI serves as a surrogate measurement for the state of the unfolded proteome. We consistently observed almost 2.5-fold increase in TMI signals specifically in LT-HSC, but not ST-HSCs or MPPs upon SYNCRIP deletion indicating that SYNCRIP is required to maintain high protein quality in HSCs. Similar results were obtained with the epichaperome probe PU-FITC, which consists of HSP90 inhibitor PU-H71 conjugated to FITC. PU-H71 selectively binds to the altered epichaperome, which reflects an accumulation of chaperon networks in an aberrant cellular stress condition. Altogether, these data further confirmed that SYNCRIP depletion tips off the proteostatic balance. To understand the molecular mechanisms underpinning the functional requirement of SYNCRIP in HSPCs, we identified 534 direct mRNA targets of SYNCRIP using hyper-TRIBE method. We performed transcriptomic and proteomic analysis of sorted LT- HSCs and LSKs respectively upon SYNCRIP deletion. We integrated these datasets and found a strong enrichment of SYNCRIP targets in control of cytoskeleton and RHO GTPase related pathways. Using immunofluorescence imaging, we confirmed that SYNCRIP deletion in HSCs resulted in 2-fold reduction in RHO GTPase CDC42 expression coupled with reduced tubulin and a loss of cellular polarity (percentage of tubulin polarized cells 56% WT vs. 40% KO). We also observed that Syncrip deficient HSCs demonstrated reduced expression of lysosomal-associated membrane protein 1 (LAMP-1) and less asymmetric distribution of LAMP1 marked lysosomes during cell division (LAMP1 asymmetric division 25% WT vs. 19% KO). Overexpression of CDC42 restored cell polarity and partly rescued ability of KO Syncrip^D/D to serially replate. Overall, SYNCRIP is required for maintenance of protein homeostasis and cell polarity of the reserve HSCs. Our study uncovers a new regulatory axis that controls stem cell stress responses to preserve HSC self-renewal.