Higher Vertebrate Specific Gene Ribonuclease Inhibitor (RNH1) Is Essential for Adult Hematopoietic Stem Cell Function and Cell Cycle Regulation

Abstract

Hematopoietic stem cells (HSC) in higher vertebrate species, especially in mammals, maintain hematopoiesis throughout adult life and require critical cell cycle regulation for their self-renewal and cell fate decisions. Although cell cycle pathways are quite conserved across animal species, it is unknown whether a higher vertebrate specific cell cycle regulation exists in adult mammalian HSCs. Recently, we have published that Ribonuclease inhibitor (RNH1) regulates erythropoiesis by controlling GATA1 mRNA translation. Here, we report that RNH1, which is present only in higher vertebrates regulates HSC cell cycle and HSC function. To study the role of RNH1 in hematopoiesis, we generated hematopoietic-specific knockout mice by backcrossing Rnh1FL/FL mice with Vav1-iCre and Mx1-Cre mice, respectively. Rnh1-deficiency (Rnh1FL/FLVav1-iCre mice) resulted in hematopoietic alterations resembling emergency myelopoiesis. At 15 weeks of age Rnh1-deficient mice had reduced hemoglobin levels (144.4 ± 2.6 vs 165.0 ± 4.2 g/L, p = 0.005), decreased lymphocytes (4.1 ± 0.8 vs 9.6 ± 1.6 K/µL, p = 0.023), increased neutrophils (3.2 ± 0.6 vs 1.5 ± 0.2 K/µL, p = 0.046) and monocytes (0.65 ± 0.05 vs 0.09 ± 0.02 K/µL, p = 0.0001) in the peripheral blood. Total bone-marrow (BM) cellularity was similar in wild type andRnh1-deficient mice, however the number of erythroid cells and lymphoid cells (T and B cells) was significantly decreased, whereas myeloid cells were significantly increased. Rnh1-deficient spleens were significantly larger than wild type controls and showed extramedullary hematopoiesis. Surprisingly, although Rnh1-deficient mice showed myeloproliferation they survived normally and did not show progression to leukemia. However, they did not tolerate even little stress, such as 35 µg LPS administration, which lead to early mortality. We analysed the progenitor populations in the BM. In line with the myelopoiesis dominant phenotype granulocyte-monocyte progenitor (GMP) cell numbers were increased but common lymphoid progenitor (CLP) and megakaryocyte-erythrocyte progenitor (MEP) cell numbers were decreased. Cell extrinsic factors such as growth factors and the bone marrow niche play a critical role in shaping lineage choice. To exclude this, we performed bone marrow transplantation experiments (BMT) by transplanting wild type (Rnh1FL/FL) and Rnh1-deficient (Rnh1FL/FLMx1-Cre+) bone marrow into lethally irradiated CD45.1 congenic mice. After reconstitution Rnh1 was deleted by administration of polyinosinic:polycytidylic acid (polyI:C). We observed a similar myelopoiesis dominant phenotype in Rnh1-deleted mice. Interestingly, we found increased numbers of long term HSCs (LT-HSCs) and short term HSCs (ST-HSCs) in Rnh1-deficient mouse BM, suggesting that RNH1 could affect HSC function. Supporting this Rnh1-deficient HSCs failed to engraft lethally irradiated mice in competitive BMT experiments. Furthermore, Rnh1-deficient HSCs produced significantly less and smaller colonies in in-vitro colony forming cell (CFC) assays. Transcriptome analysis showed increased expression of genes related to cell cycle, kinetochore, DNA damage and decreased expression of genes related to stem cell function in Rnh1-deficient LT-HSCs and ST-HSCs. Corroborating this, Rnh1-deficient LT-HSCs and ST-HSCs showed increased S/G2/M phase in cell cycle analysis. In line with this, at the molecular level, we found that RNH1 directly binds to cell-cycle related proteins such as cyclin-dependent kinase 1 (CDK1), cell-division cycle protein 20 (CDC20) and mitotic checkpoint protein BUB3, suggesting direct involvement of RNH1 in cell cycle regulation. Confirming this, pharmacological inhibition of CDK1 (RO-3306, 10 µM) in Rnh1-deficinet ST-HSCs restored colony size in CFC assays, suggesting that RNH1 and CDK1 inhibition have a synergistic effect in ST-HSCs. In summary, our results demonstrate that RNH1, which is present only in higher vertebrates, is essential for HSC cell cycle regulation and steady state hematopoiesis. Disclosures No relevant conflicts of interest to declare.

CITED2 coordinates key hematopoietic regulatory pathways to maintain the HSC pool in both steady-state hematopoiesis and transplantation

“…However, ROS levels were not elevated in Cited2 CKO HSCs ( Figure S3 B), and treatment of Cited2 CKO mice with the antioxidant N-acetyl-L-cysteine (NAC) did not rescue HSC depletion ( Figures S3 C and S3D), suggesting that ROS is unlikely to be responsible for the observed effects in HSCs lacking Cited2 . Furthermore, in addition to the GSEA, our data revealed that several genes essential for HSC functions, including Prdm16 , Men1 , Rnh1 , and Akt2 were downregulated in Cited2 CKO HSCs ( Figure S4 ) ( Andina et al., 2019 ; Gudmundsson et al., 2020 ; Juntilla et al., 2010 ; Maillard et al., 2009 ). Finally, further interrogation of promoters of the downregulated genes revealed the presence of motifs for STAT3, STAT4, E2A, and ZFX transcription factors ( Figure 5 F), all of which are required for HSC maintenance and function ( Galan-Caridad et al., 2007 ; Holmfeldt et al., 2016 ; Mantel et al., 2012 ; Semerad et al., 2009 ).…”

Ribonuclease inhibitor and angiogenin system regulates cell type–specific global translation

“…Further, knockdown of RNH1 in human CD34 + cells decreased erythroid differentiation ( 20 ). Similarly, deletion of RNH1 in mouse hematopoietic cells was found to dysregulate hematopoiesis and cause anemia ( 48 ). Moreover, mutations in the RNH1 gene in human have also been associated with anemia phenotype ( 21 ).…”

Ribonuclease Inhibitor and Angiogenin collaboratively regulate cell-type-specific global translation

“…Further, knockdown of RNH1 in human CD34 + cells decreased erythroid differentiation 21 . Similarly, deletion of RNH1 in mouse hematopoietic cells (HSCs) was found to dysregulate hematopoiesis and cause anemia 45 . Moreover, mutations in the RNH1 gene in human have also been associated with anemia phenotype 22 .…”