Trmt6 loss leads to phenotypic HSC expansion and Long-term repopulation capacity decline
Before investigating the biological roles of Trmt6 and Trmt61a in haematopoietic system, we initially assessed the expression of Trmt6 and Trmt61a genes across multiple haematopoietic cell populations utilizing publicly available single-cell transcriptomic data47. Trmt6 and Trmt61a are broadly expressed in haematopoietic system (Figs. S1a and g). Notably, we observed a consistent upregulation of Trmt6 but not Trmt61a in donor-derived bone marrow cells derived several days post-transplantation of HSCs47 (Figs. S1b and h). Treatment of HSCs with 5-Fu48, ionizing radiation49, or LPS50 resulted in upregulation of Trmt6 but not Trmt61a at 1-3 days (Figs. S1c–S1e and S1i–S1k), while in aged HSCs the expression of Trmt6 slightly decreased51 (Fig. S1f), indicating that Trmt6 and Trmt61a may play a role in homeostasis and regeneration of HSCs.
To understand the functional role of Trmt6 in haematopoietic system, we generated Mx1-Cre;Trmt6fl/fl (Trmt6-/-) mice and confirmed the deletion efficiency of Trmt6 (Figs. 1a, b and S2a). Although the size and weight of Trmt6-/- spleen were comparable with that of wild-type mice, a reduction in frequency of B lymphocytes and an increase in the frequency of myeloid cells were observed in spleen of Trmt6-/- mice (Figs. S2b–d). In peripheral blood (PB) of Trmt6-/- mice, the reduction in frequency of B lymphocytes and the increase in the frequency of myeloid and T cells were more noticeable (Fig. S2e). Furthermore, complete blood counts revealed a significant decrease in the number of white blood cells and lymphocytes and a significant increase in the number of platelets and neutrophils in Trmt6-/- mice (Figs. S2f–i). Those results suggested an abnormal lineage output in Trmt6-/- mice.
a Targeting strategy to generate the Trmt6 conditional knockout (cKO) mouse. b Experimental scheme for Trmt6-/- and Trmt6+/+ hematopoiesis analysis. c FACS analysis of LT-HSCs (CD150+CD48–FlK2–Lin–c-Kit+Sca-1+), ST-HSCs (CD150–CD48–FlK2–Lin–c-Kit+Sca-1+), and MPPs in Trmt6-/- and Trmt6+/+ BM cells. Representative FACS profiles are shown. n = 5 mice per genotype. d Frequency of LT, ST, MPP2, MPP3 and MPP4 in BM cells are shown. n = 5 mice per genotype. e Frequency of MEP, GMP and CMP in BM cells are shown. n = 5 mice per genotype. f Cell cycle analysis of Trmt6-/- and Trmt6+/+ LT-HSCs. Representative FACS profiles of Ki67 antibody and DAPI staining are shown. g Frequency of the cell cycle distribution in Trmt6-/- and Trmt6+/+ LT-HSCs are shown. n = 5 mice per genotype. h Experimental schematic for serial competitive transplantation with Trmt6-/- and Trmt6+/+ BM cells. i Percentage of donor-derived PB cells at the indicated time points in the first competitive transplantation. n = 6 mice per genotype. j Percentage of donor-derived LT-HSCs 20 weeks after first transplantation. n = 6 mice per group. k Percentage of donor-derived PB cells at the indicated time points in the second competitive transplantation. n = 6 mice per group. l Percentage of donor-derived LT-HSCs 16 weeks after secondary transplantation. n = 6 mice per group. Data represent the mean ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. For all the above statistics, P values were obtained using unpaired parametric two-tailed t-test. Exact P values are provided as Source Data.
To formally and functionally investigate the effects of Trmt6 loss in the haematopoietic system, we evaluated the immunophenotypic composition of the HSPC compartments in cohorts of Trmt6+/+ and Trmt6-/- mice. Flow cytometry analysis showed a ~ 4-fold increase in the frequency of CD150+CD48–FlK2– LSK cells (LT-HSCs) and progenitors (MPP, GMP, CMP, and MEP) in Trmt6-/- bone marrow at 1week after polyI:C (Fig. 1c–e). Taken together, Trmt6 deletion causes a dramatic increase in the numbers of HSCs.
To acquire further insight into the expansion of HSC pools after Trmt6 deletion, we conducted cell cycle analysis on LT-HSCs and LSK cells. Ki67 staining revealed a significantly lower frequency of G0 cells (Ki67–) in Trmt6–/- LT-HSCs and LSK cells than that in Trmt6+/+ cells (80% in Trmt6+/+ vs. 58% in Trmt6–/- LT-HSCs) (Figs. 1f, g and S3a). Cycling HSCs are more sensitive to 5-Fluorouracil (5-Fu) cytotoxicity, while quiescent HSCs remain viable after 5-Fu treatment52. Trmt6–/- mice challenged with sequential 5-Fu treatment died significantly earlier than the Trmt6+/+ controls (Figs. S3b and c), indicating that Trmt6-null HSCs were less quiescent.
Aberrantly enhanced proliferation often consequentially exhausts HSCs and affects their function in haematopoietic reconstitution8,9,24,53,54,55. A significant reduction of BM cells in Trmt6–/- mice was found by examining the total number of bone marrow cells (Fig. S3d). To further observe the HSC depletion phenomenon caused by TRMT6 deficiency, flow cytometry analysis showed that the frequency of LT-hematopoietic stem and progenitor cells (MPP2, MPP3, MPP4) in Trmt6–/- bone marrow was significantly decreased 4 weeks after injection of PolyI:C (Fig. S3e), and at the same time, compared with control mice, the survival rate of Trmt6–/- mice was significantly decreased (Fig. S3h), and even less for stress induced by 5-Fu (Figs. S3b and c) and 4 Gy irradiation (Figs. S3i and j). Collectively, these data suggest that Trmt6 depletion has a significant impact on hematopoiesis.
We hypothesize that although higher in number, the functionality of Trmt6-null HSCs might be exhausted faster upon transplantation or stresses. To this end, we conducted HSC competitive transplantation experiments. Surprisingly, in comparison with Trmt6+/+ HSCs, Trmt6–/- HSCs exhibited a ~ 10-fold reduction in their long-term repopulating ability followed by PB and LT-HSC chimerism analysis (Figs. S3f and g).
To exclude the potential impact of TRMT6 deficiency on HSC homing upon transplantation, Trmt6+/+ Mx1-Cre or Trmt6fl/fl Mx1-Cre BM cells were first transplanted without inducing Trmt6 deletion. The recipient mice were then injected with polyI:C at 6 weeks after transplantation to induce Trmt6 deletion, followed by PB chimerism analysis and secondary transplantation (Fig. 1h). To this end, we conducted competitive transplantation experiments by injecting 1 × 106 Trmt6+/+ or Trmt6–/- BM cells (CD45.2 background) into lethally irradiated congenic-recipient mice (CD45.1) along with 1 × 106 competitive BM cells (CD45.1), followed by secondary transplantation (Fig. 1h). In contrast to Trmt6+/+ cells, which gave rise to stable long-term multi-lineage reconstitution in the recipient mice, Trmt6–/- LT-HSCs and PB exhibited a ~ 10-fold reduction in their long-term repopulation ability at 20 weeks post transplantation (Figs. 1i, j and S3k–m). The analysis of recipient mice revealed a significant decrease in the frequency of donor-derived PB and LT-HSC cells 16 weeks after the secondary transplantation (Figs. 1k, l and S3n–p), suggesting that Trmt6 deletion produces a cell-autonomous functional defect in HSCs. Altogether, these data indicate that TRMT6 depletion impaired the long-term multi-lineage repopulation activity and self-renewal capacity of HSCs in a cell intrinsic manner.
Single-cell RNA sequencing (scRNA-seq) revealed an activated mTORC1 signaling pathway in Trmt6 deficient HSC populations
To elucidate the molecular mechanism of Trmt6 in regulating haematopoiesis, we performed single-cell RNA sequencing (scRNA-seq) on sorted HSPCs (LSK) cells isolated from bone marrow (BM) of young adult Trmt6+/+ and Trmt6-/- mice (Fig. 2a and S2j). In total, 8258 out of 8869 and 8599 out of 9260 cells from Trmt6+/+ and Trmt6-/- BM passed quality control, respectively. Cells were annotated using SingleR package by comparing transcriptome between our and Nestorowa et al. data56 (Figs. 2b, c and S4a). Procr/Pdzk1ip1, Myct1, Dntt, Ctsg and Car1 were specifically and highly expressed genes in the predicted long-term HSCs (LT-HSCs), MPPs, LMPPs, CMPs, GMPs and megakaryocyte-erythroid progenitors (MEPs), respectively (Figs. S4b and c). Dramatic changes of cell type composition were observed in Trmt6-/- HSPCs, specifically, the fraction of LT-HSCs increased, along with increased fraction of multipotent progenitor 2 (MPP2) population and decreased fraction of other cell types (Fig. 2d). Trmt6-/- HSPCs exhibited higher proliferation score, and the percentage of cells with S and G2/M cell cycle signature elevated in Trmt6-/- HSCs and MPPs (Figs. 2e, f), suggesting a disturbed quiescence-proliferation transition in Trmt6-/- HSCs.
a Experimental schematic for the generation of mice with bone marrow specific deletion of Trmt6 and the obtaining of HSPCs for scRNA-seq. Trmt6fl/+ mice were crossed with interferon-inducible transgenic Mx1-Cre mice to generate Trmt6+/+ Mx1-Cre and Trmt6fl/fl Mx1-Cre mice. Mice were then treated with intraperitoneal injections of 300 ug/kg pIpC every other day seven times to delete the Trmt6 alleles. ScRNA-seq was performed for FACS-purified HSPCs. b UMAP plots showing nine populations of Trmt6+/+ HSPCs and Trmt6-/- HSPCs annotated with Nestorowa et al. data. Colors indicate cell types. c Histograms showing the compositions of nine populations. Colors indicate cell types. d Boxplot comparing the proliferation score of cells in each population between Trmt6+/+ HSPCs and Trmt6-/- HSPCs. The standard boxplot notation was used (lower/upper hinges-first/third quartiles; whiskers extend from the hinges to the largest/smallest values no further than 1.5 x inter-quartile ranges; middle line-the median). The differences are tested by two-sided Wilcoxon rank sum test, *, P < 0.05; **, P < 0.01; ***, P < 0.001. e Histograms showing the proportions of nine populations in each cell cycle stages between Trmt6+/+ and Trmt6-/-. f GSEA analyzes for genes affected in the LT-HSCs of Trmt6-/- versus Trmt6+/+ control mice showing positive enrichment of proliferation signature (orange) and negative enrichment of quiescence signatures (blue). NES, normalized enrichment score. g Dot plot GSEA analyzes for genes affected in nine populations of Trmt6-/- versus Trmt6+/+ to test enrichment for enrichment of hallmarks. h GSEA analyzes for genes affected in the LT-HSCs of Trmt6-/- versus Trmt6+/+ showing positive enrichment of mTORC1 signaling. Heatmap shows the scaled expression of leading-edge subset of genes in mTORC1-signaling. i Jitter plots and density plots showing the distribution of pseudotime predicted by Monocle3 for nine populations. Colors indicate cell types. j Dot plot showing changes of the ratio between myeloid- and lymphoid-biased HSPCs after Trmt6 deletion. The upward and downward arrows indicate higher or lower ration between myeloid- and lymphoid-biased HSPCs after Trmt6 deletion, respectively. P values in d and f-h were BH-adjusted. Exact P values are provided as Source Data.
To explore the mechanism whereby Trmt6 controls the quiescence-proliferation transition, we performed differential expression analysis for each cell type. In each cell type except for MEPs (where only 3 genes were dysregulated), hundreds of genes showed dysregulation following Trmt6 depletion (adjusted P < 0.05 and |log2FC | > 0.25), with some of them exhibiting a larger change in expression (adjusted P < 0.05 and |log2FC | > 1) (Fig. S4d). In the case of LT-HSCs, 411 genes were found to be differentially expressed upon Trmt6 depletion, with 325 genes upregulated and 86 genes downregulated. Out of these, 36 genes were specifically differentially expressed in LT-HSCs, while the remaining 375 genes showed dysregulation in downstream haematopoietic cells. This observation suggested that the impact of Trmt6 depletion is long-lasting. Gene set enrichment analysis (GSEA) of gene expression changes in Trmt6-/- LT-HSCs showed upregulated proliferation signature and downregulated quiescence signature (Fig. 2g). Cell-cell communication analysis showed that HSCs interacted more frequently with others in Trmt6-/- mice than those in Trmt6+/+ mice (Figs. S4e and f). Above results indicated Trmt6 depletion, to some extent, deprived the restriction of quiescence-proliferation transition. Further GSEA reveals more active expression of several hallmark gene sets Trmt6-/- HSPCs, including mTORC1 signalling, unfolded protein response, MYC targets, oxidative phosphorylation, and glycolysis (Fig. 2g). Additional gene set variation analysis (GSVA) also showed significantly higher expression of mTORC1 signalling genes in Trmt6-/- LT-HSCs than that in Trmt6+/+ LT-HSCs (Wilcoxon rank sum test, P < 2.2e-16) (Fig. 2h). It has long been established that mTORC1 signalling governs the quiescence of HSCs57,58. therefore, Trmt6 likely controls the quiescence-proliferation transition through regulating mTORC1 signalling. Consistent with the observed lineage-bias of haematopoietic system in Trmt6-/- mice and the known role of mTOR signalling pathway as the central hub for HSC fate coordination27, pseudotime analysis showed a prone-to-myeloid transcriptomic change of HSCs in Trmt6-/- mice (Fig. 2i), and the transcriptomic shift leading to lineage-bias likely happened in early differentiation stage, namely, in LT-HSCs (Fig. 2j). In all, single-cell transcriptomic profiling of HSPC populations supports a prominent role for Trmt6 in HSC quiescence.
tRNA-m1A58 modification is required for efficient Tsc1 mRNA translation and mTORC1 signaling regulation
Previous studies have shown that the mammalian target of rapamycin complex 1 (mTORC1) is critical for cell metabolism, cell growth and cell cycle progression in HSCs. Therefore, we first examined the activity of mTORC1. We found that the phosphorylation levels of ribosomal protein S6 (p-S6) were significantly increased in Trmt6–/- LT-HSCs and LSK cells compared with those in Trmt6+/+ LT-HSCs and LSK cells (Fig. 3e and S5a). Trmt6-/- HSCs also had other features of mTORC1 hyperactivation, such as increased cell size (Fig. S5b), increased ROS (Fig. S5c) and peak mitochondrial membrane potential changes (Fig. S5d). Although the apoptotic proportion of HSCs did not change significantly, the apoptotic rate of Lin+ cells was high (Fig. S5e). It is plausible that TRMT6 loss resulted in sustained mTORC1 activation, which was responsible for the phenotypic expansion and functional decline of HSCs.
a LC-MS/MS quantification of m1A levels in the RNA ( >200 nt) and tRNA ( >50nt and <200 nt) purified from Trmt6-/- and Trmt6+/+ HSPC, presented as percentage of unmodified A. Trmt6+/+ HSPC RNA ( >200 nt), n = 4 independent biosamples and tRNA, n = 5 independent biosamples. Trmt6-/- HSPC RNA ( >200nt), n = 5 independent biosamples and tRNA, n = 4 independent biosamples. b Global m1A levels were detected in tRNA ( >50nt and <200 nt) purified from Trmt6-/- and Trmt6+/+ HSPC using dot blot assay. Data were repeated five times. c The decrease in magnitude (the tRNA-m1A58 level in Trmt6-/- HSPCs minus the level in Trmt6+/+ HSPCs) of the tRNA-m1A58 level in each tRNA after TRMT6 deletion. d The position of codon GCA (corresponding to tRNA-Ala-TGC) in mouse TSC1 protein. e Flow cytometry analysis of p-S6 levels in the LT-HSCs of Trmt6-/- and Trmt6+/+ mice. Median fluorescence intensity of p-S6 in Trmt6-/- (n = 3 mice) and Trmt6+/+ (n = 4 mice) LT-HSCs. f The relative mRNA expression levels of genes encoding LKB1, AMPK, TSC1, GSK3β, REDD1 and RHEB in Trmt6-/- LT-HSCs vs. Trmt6+/+ LT-HSCs are shown. n = 4 independent biosamples. g Flow cytometry analysis of TSC1 levels in the LT-HSCs of Trmt6-/- and Trmt6+/+ mice. Median fluorescence intensity of TSC1 in Trmt6-/- (n = 3 mice) and Trmt6+/+ (n = 4 mice) LT-HSCs. h Ribosome occupancy of LKB1, AMPK, TSC1, GSK3β, REDD1, RHEB and control mRNAs was measured by RT-PCR as the relative expression ratio (RER) of polyribosome mRNAs to the input mRNAs after sucrose gradient fractionation of polyribosomes. Data represent the mean ± SD from three independent experiments. *, P < 0.05; **, P < 0.01; ***, P < 0.001. For all the above statistics, P values were obtained using unpaired parametric two-tailed t-test. Exact P values are provided as Source Data.
m1A at position 58 (m1A58) in tRNA can be catalyzed by a methyltransferase complex, containing an RNA binding component TRMT6 and a catalytic component TRMT61A34,44. To further determine whether TRMT6 mediated methyltransferase complex exerted function through its enzymatic activity, we use quantitative mass spectrometry (LC-MS/MS) and RNA dot blot assay to analysis m1A signals of big RNA ( > 200 nt RNA) and small RNA ( >50 nt and <200 nt RNA, mostly tRNA). We found that only global tRNA-m1A58 modification levels in Trmt6–/- HSPCs were decreased compared with those of Trmt6+/+ HSPCs (Figs. 3a, b).
tRNA-m1A58 modification is required for efficient mRNA translation during initiation and elongation45,59. RT-PCR analysis of targets protein antibody found that the mRNA levels of the upstream targets (LKB1, AMPK, TSC1, GSK3β, REDD1, RHEB) of mTORC1 pathways30 did not differ (Fig. 3f). Furthermore, FACS staining of target protein antibody further confirmed that the protein levels of the upstream targets (LKB1, AMPK, GSK3β, REDD1 and RHEB) of mTORC1 pathways did not differ (Fig. S5f), but TSC1 were significantly down-regulated in Trmt6-null LT-HSCs and LSK cells (Fig. 3g, S5g and S5h). To establish whether Tsc1 mRNA, through its codon content, requires tRNA-m1A58 modification during translation, we performed polyribosome RT-PCR experiments to quantify the ribosome occupancy of Tsc1 mRNAs. Notably, we found a dramatic decrease in the accumulation of ribosomes on Tsc1 mRNAs but not on LKB1, AMPK, GSK3β, REDD1, RHEB or control transcripts upon TRMT6 depletion, suggesting that tRNA-m1A58 is more likely to regulate the translation elongation of Tsc1 mRNA (Fig. 3h).
TSC1 deficiency leads to reduced reconstruction capacity of HSCs. To further explore the potential molecular mechanisms of TSC1 translational enhancement by tRNA-m1A58 modification, we analyzed the tRNA-m1A sequencing data of WT versus Trmt6–/- HSPCs to determine the magnitude of the reduction in m1A58 modification levels on each tRNA after TRMT6 deletion (Figs. S6a–c). We found that the m1A58 levels on most tRNAs were reduced to varying degrees after TRMT6 deletion, and the tRNAs decoding Alanine was among the most affected tRNAs (Fig. 3c). Alanine was also among the codons high frequently used by Tsc1 mRNA (Fig. 3d and S6e). To further identify the transcripts whose translation were regulated by these m1A58 elevated tRNAs, we performed ribosome profiling sequencing (Ribo-seq) analysis in TRMT6 depleted and control HSPCs. With Ribo-seq data, we found Tsc1 gene has lower translation efficiency in Trmt6–/- HSPCs (Fig. S6f).
To determine whether TSC1 translation deficiency was due to mRNA translation initiation or elongation in Trmt6–/- HSPCs, we analyzed the m1A58 levels and expression levels of initiator-methionine tRNA and found that neither was decreased in Trmt6–/- HSPCs (Fig. 3c, S6b and S6d), suggesting that tRNA-m1A58 is more likely to regulate the translation elongation of Tsc1 mRNA. Taken together, these results demonstrate that tRNA-m1A58 is essential for efficient Tsc1 mRNA translation in HSPCs.
To further explore the potential molecular mechanisms of Tsc1 translational enhancement by tRNA-m1A58 modification, we reanalyzed the tRNA-m1A sequencing data of WT versus Trmt6–/- HSPCs to determine the magnitude of the reduction in m1A58 modification levels on each tRNA after TRMT6 deletion. Ala was among the codons most frequently used by Tsc1 mRNA; thus, we remodeled Tsc1 cDNA such that the most abundant Alanine codons were replaced by their synonymous codons decoded by tRNAs least affected by TRMT6 deletion (Fig. S7a). Accordingly, the GCA/GCG (Ala) codons were replaced by GCC (Ala) (Fig. S7a). Expression of this mutant Tsc1 via lentivirus in WT and Trmt6-/- HSPCs (Fig. S7b) was sufficient to rescue the defective expression of TSC1 (Fig. S7c) and single clone formation in Trmt6-/- HSCs in vitro (Fig. S7d), confirming that tRNA-m1A58 modification directly regulates Tsc1 mRNA translation through codon decoding.
Furthermore, we knockdowned the expression of TRMT61A in HSCs, an essential m1A catalytic partner for TRMT6 in the m1A ‘writer’ complex (Fig. S8a). HSCs of sh-Trmt61a showed a similar phenotype to Trmt6-KO HSCs (Figs. S8b–g). Specifically, using the above methods, we found a similar self-renewal defect in HSCs of sh-Trmt61 (Figs. S8c and d). Thus, our data demonstrate that Trmt61a-deficiency induced mTORC1 activation is due to the depletion of tRNA-m1A58 modification.
Inhibition of mTOR pathway ameliorates aberrant proliferation and self-renewal defects of Trmt6 knockout HSC
To examine the relationship between the enhanced mTOR signaling and dysregulated haematopoiesis in Trmt6–/- mice, we treated Trmt6–/- mice and age-matched control mice with rapamycin for 3 weeks (Fig. 4a). Importantly, treatment with rapamycin recovered the number of LT-HSCs, ST-HSCs, MPP2, MPP3 and MPP4 as well as B, T and myeloid cells of PB or spleen in Trmt6–/- mice compared with that in Trmt6+/+ mice (Fig. 4b, c and S9a–c). Treatment with rapamycin recovered the cell cycle defects of LT-HSCs and LSK cells in Trmt6–/- mice compared with that in Trmt6+/+ mice (Fig. 4d and S9d). Furthermore, there is a decrease in p-S6 levels in Trmt6-/- mouse after rapamycin treatment compared to vehicle, but no changes in Trmt6+/+ mouse, suggesting that rapamycin treatment partially rescued the overactivation of mTOR signaling (Fig. 4e and S9e).
a The experimental design for treatment with an mTOR inhibitor rapamycin in Trmt6-/- and Trmt6+/+ mice. b FACS analysis of LT-HSCs, ST-HSCs and MPPs in Trmt6-/- and Trmt6+/+ BM cells after mTOR inhibitor rapamycin treatment in vivo. Representative FACS profiles are shown. n = 5 mice per genotype. c Frequency of LT-HSCs in BM cells are shown after mTOR inhibitor rapamycin treatment in vivo. n = 5 mice per genotype. d Experimental schematic for serial competitive transplantation with Trmt6-/- and Trmt6+/+ BM cells after mTOR inhibitor rapamycin treatment in vivo. e Flow cytometry analysis of p-S6 levels in the LT-HSCs of Trmt6-/- and Trmt6+/+ mice after mTOR inhibitor rapamycin treatment in vivo. Median fluorescence intensity of p-S6 in Trmt6-/- and Trmt6+/+ LT-HSCs after mTOR inhibitor rapamycin treatment in vivo. n = 3 mice per genotype. f The experimental design for transplantation using whole bone marrow cells treated with rapamycin. Whole bone marrow cells isolated from Trmt6-/- and Trmt6+/+ mice that had been treated with rapamycin for 3 weeks were transplanted into lethally irradiated recipient mice with competitor cells. Recipient mice were continuously treated with rapamycin after transplantation (results in Fig. 4g–h). g After transplantation, the frequency of donor-derived cells in peripheral blood were analyzed. n = 5 mice per genotype. h After transplantation, the frequency of donor-derived cells in LT-HSCs were analyzed. n = 5 mice per genotype. Data represent the mean ± SD from three independent experiments. ns, P value ≥ 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001. For all the above statistics, P values were obtained using one-way ANOVA followed Dunnett’s multiple comparisons test. Exact P values are provided as Source Data.
To test if TSC1 and TRMT6 or enzyme-dead TRMT6 overexpression can rescue the defect in vivo, Trmt6-/- and Trmt6+/+ HSCs from primary mice were transduced with TSC1, TRMT6 or enzyme-dead TRMT6 (TRMT6mut) overexpression (OE) lentivirus, and transduced cells were transplanted into CD45.1 recipients (Figs. S10a and d). Chimerism in the bone marrow was evaluated at 16 weeks post-transplantation to read out long-term engraftment. We observed that OE of TSC1 can partially rescued the function of Trmt6-deficient HSCs (Figs. S10b and c). However, TRMT6mut OE did not improve Trmt6-/- HSCs’ engraftment deficiency at the level of HSC and PB (Figs. S10e and f). The data suggested that TRMT6 play roles by the function of the m1A enzyme in HSCs, while TSC1 at least in part is responsible for TRMT6’s role in maintaining reconstitution in HSCs
To investigate the effect of mTORC1 inhibition on the functionality of Trmt6–/- HSCs in vivo, we conducted competitive transplantation assays (Fig. 4f) and found that rapamycin treatment partially rescued the reconstitution ability of Trmt6–/- donor-derived HSCs in the PB and LT-HSC compartment of the recipients compared with vehicle treatment, indicating that mTORC1 inhibition also rescued the function of Trmt6-deficient HSCs (Figs. 4g, h). Taken together, these data suggest that overactive mTORC1 pathway partially causes dysregulated haematopoiesis in Trmt6–/- mice.
- SEO Powered Content & PR Distribution. Get Amplified Today.
- PlatoData.Network Vertical Generative Ai. Empower Yourself. Access Here.
- PlatoAiStream. Web3 Intelligence. Knowledge Amplified. Access Here.
- PlatoESG. Carbon, CleanTech, Energy, Environment, Solar, Waste Management. Access Here.
- PlatoHealth. Biotech and Clinical Trials Intelligence. Access Here.
- Source: https://www.nature.com/articles/s41467-024-50110-9