SNORD113-114 Cluster Maintains Hematopoietic Stem Cell Self-Renewal Via Orchestrating the Translation Machinery

Hematopoietic stem cells (HSCs) self-renew and differentiate to replenish the pool of blood cells, which require a low but finely-tuned protein synthesis rate. Nonetheless, the translatome landscape in HSCs and how the translation machinery influences HSC self-renewal remain largely elusive. To systematically profile the translatome of hematopoietic cells and identify cell type-specifically translated genes, we initially sorted LT-HSCs (Lin^-Sca-1⁺c-Kit⁺CD34^-Flk2^-), multipotent progenitors (MPP, Lin^-Sca-1⁺c-Kit⁺CD34⁺Flk2⁺), committed progenitor cells (CLPs, CMPs, GMPs and MEPs) and mature lineage cells (myeloid cells, B cells, T cells and erythroid cells) using fluorescent activated cell sorting (FACS). Subsequently, we conducted ultra-low-input Ribo-seq (160 cells/sample) and generated fingerprint of translatome of all hematopoietic cells. Unsupervised hierarchical clustering generated fingerprints of signature genes of each cell type, suggesting their involvement in specific cell fate determination. Furthermore, GO term analysis using Hallmark pathways revealed featured pathways that were enriched in specific cell types. The pathways enriched in HSCs were related to rRNA processing, cell adhesion, cell cycle, WNT signaling, and so on. Compared with MPPs, translationally up-regulated genes in HSCs (n=676) were enriched in pathways including snoRNA binding, definitive hemopoiesis, and snoRNP complex, indicating the crucial role of snoRNAs in maintaining HSC homeostasis.

Following the clue, we then systematically profiled the snoRNA expression in hematopoietic stem and progenitor cells, committed progenitor cells and mature lineage cells. We discovered that snoRNAs belonging to the SNORD113-114 cluster were predominantly enriched in LT-HSCs, and their expression decreased dramatically along with HSC differentiation, implying their crucial role in HSC homeostasis. Furthermore, maternal knockout of this cluster (Mat-KO) significantly impairs HSC in vitro and in vivo self-renewal ability, while loss of the paternal allele shows no obvious phenotype. Cell cycle profiles of HSCs in Mat-KO mice presented significant decrease in G0 phase and increase in the G1 phase fraction, indicating cell cycle dysregulation in the absence of the SNORD113-114 cluster. Transcriptome analysis using bulk RNA-seq and single-cell RNA-seq also revealed dysregulations in translation machinery and cell cycle regulation. Besides, translatome profiling of WT/Mat-KO HSCs revealed dysregulation of genes enriched in cell cycle, rRNA processing, ribosome biogenesis pathways.

Mechanistically, Loss of SNORD113-114 cluster resulted in down-regulation of 2’-O-Methylation at multiple sites of rRNA, as proved by LC-MS and RiboMethSeq. rRNA processing was also disturbed, as indicated by decrease in 46s and 32s pre-rRNA and abnormal accumulation of 34s pre-rRNA, which finally dampened assembly of 60S ribosome subunit. Accordingly, translation function of ribosome was also undermined.The dysregulation of translation machinery induced nucleolar stress in HSCs, which exempted p53 from MDM2 mediated proteasomal degradation and eventually led to apoptosis. Finally, overexpression of snoRNAs or L-leucine mediated translation recovery rescues deficiency in Mat-KO HSCs. Collectively, our study provides a new facet to our understanding of snoRNA-mediated regulation in HSC homeostasis.