RNA binding protein SYNCRIP maintains proteostasis and self-renewal of hematopoietic stem and progenitor cells

Tissue homeostasis is maintained after stress by engaging and activating the hematopoietic stem and progenitor compartments in the blood. Hematopoietic stem cells (HSCs) are essential for long-term repopulation after secondary transplantation. Here, using a conditional knockout mouse model, we revealed that the RNA-binding protein SYNCRIP is required for maintenance of blood homeostasis especially after regenerative stress due to defects in HSCs and progenitors. Mechanistically, we find that SYNCRIP loss results in a failure to maintain proteome homeostasis that is essential for HSC maintenance. SYNCRIP depletion results in increased protein synthesis, a dysregulated epichaperome, an accumulation of misfolded proteins and induces endoplasmic reticulum stress. Additionally, we find that SYNCRIP is required for translation of CDC42 RHO-GTPase, and loss of SYNCRIP results in defects in polarity, asymmetric segregation, and dilution of unfolded proteins. Forced expression of CDC42 recovers polarity and in vitro replating activities of HSCs. Taken together, we uncovered a post-transcriptional regulatory program that safeguards HSC self-renewal capacity and blood homeostasis.

File Name: Supplementary Data 2 Description: scRNA-seq cluster vs rest DEG. Differentially expressed genes between a cluster vs. the rest of other clusters characterized in scRNA-seq datasets. Total 21 clusters as defined in cluster assignment. The statistical test was performed using DEseq Bioconductor package. In general, DEseq algorithm performs estimation of size factors and estimation of dispersion prior to negative binomial GLM fitting and Wald statistic. A one-sided Wilcoxon test was performed to determine the statistical significance between the log2 fold changes (log2FC).
File Name: Supplementary Data 3 Description: scRNA-seq HSC C1 vs HSC C2 pval rank list. The analysis and statistical test were performed using https://maayanlab.cloud/Enrichr/. In brief, Enrichr analysis includes three enrichment scores based on three statistical tests: 1) the Fisher exact test; 2) the z-score calculating deviation from the expected rank by the Fisher exact test; and 3) a combined score which is the multiplication of the log p value obtained from the test 1 and test 2.
File Name: Supplementary Data 7 Description: Enrichr analysis of HSC C1 KO vs WT. Table showing results of gene set enrichment analysis using https://maayanlab.cloud/Enrichr/ of differentially expressed genes (FDR <=0.05; up and down regulated genes) within cluster HSC C1 between Syncrip f/f (n=3) and Syncrip ∆/∆ (n=3). Pathway analysis on GO biological process 2021 and Reactome 2016. The analysis and statistical test were performed using https://maayanlab.cloud/Enrichr/. In brief, Enrichr analysis includes three enrichment scores based on three statistical tests: 1) the Fisher exact test; 2) the z-score calculating deviation from the expected rank by the Fisher exact test; and 3) a combined score which is the multiplication of the log p value obtained from the test 1 and test 2.  Table showing significant edited sites identified with HyperTRIBE in HSCs and MPPs (fpkm > 5, p-adj > 0.05, differential edit frequency > 0.1) To define difference in editing frequencies, we used betabinomidal distribution and p values were were adjusted to control for false discovery rate (FDR) using a Benjamin-Hochberg correction. The statistical computation was performed using R packages VGAM (Version 1.1-2) and bbmle (Version 1.0.23.1) as described in Nguyen DTT, et al. 2020 File Name: Supplementary Data 10 Description: SYNCRIP direct targets in HSCs and MPPs.  Reactome, 2016, andKEGG 2021. For directionality, proteomics results were used (p-value <0.1, Log2FC ≤ -1). The analysis and statistical test were performed by https://maayanlab.cloud/Enrichr/. In brief, Enrichr analysis includes three enrichment scores based on three statistical tests: 1) the Fisher exact test; 2) the z-score calculating deviation from the expected rank by the Fisher exact test; and 3) a combined score which is the multiplication of the log p value obtained from File Name: Supplementary Movie 1 Description: Representative videos for CDC42 Rescue in LSK cells described in Figure 6S-T. Videos show GFP expression in green, tubulin in red, CDC42 in magenta and DAPI counterstain in blue. Only cells with GFP integrated intensities > 100000 were used for quantification in Figure 6T. Movie 1 represents Syncrip f/f LSK cells that were transduced with empty vector (EV) MIGR1 retrovirus.
File Name: Supplementary Movie 2 Description: Representative videos for CDC42 Rescue in LSK cells described in Figure 6S-T. Videos show GFP expression in green, tubulin in red, CDC42 in magenta and DAPI counterstain in blue. Only cells with GFP integrated intensities > 100000 were used for quantification in Figure 6T. Movie 2 represents Syncrip / LSK cells that were transduced with empty vector (EV) MIGR1 retrovirus.
File Name: Supplementary Movie 3 Description: Representative videos for CDC42 Rescue in LSK cells described in Figure 6S-T. Videos show GFP expression in green, tubulin in red, CDC42 in magenta and DAPI counterstain in blue. Only cells with GFP integrated intensities > 100000 were used for quantification in Figure 6T. Movie 3 represents Syncrip / LSK that were transduced with CDC42 Overexpression (OV) retrovirus.
File Name: Supplementary Movie 4 Description: Representative videos for CDC42 Rescue in LSK cells described in Figure 6S-T. Videos show GFP expression in green, tubulin in red, CDC42 in magenta and DAPI counterstain in blue. Only cells with GFP integrated intensities > 100000 were used for quantification in Figure 6T. Movie 4 represents Syncrip f/f LSKs that were transduced with CDC42 Overexpression (OV) retrovirus.