Abstract
RNA modifications are emerging as key determinants of gene expression. However, compelling genetic demonstrations of their relevance to human disease are lacking. Here, we link ribosomal RNA 2′-O-methylation (2′-O-Me) to the etiology of dyskeratosis congenita. We identify nucleophosmin (NPM1) as an essential regulator of 2′-O-Me on rRNA by directly binding C/D box small nucleolar RNAs, thereby modulating translation. We demonstrate the importance of 2′-O-Me-regulated translation for cellular growth, differentiation and hematopoietic stem cell maintenance, and show that Npm1 inactivation in adult hematopoietic stem cells results in bone marrow failure. We identify NPM1 germline mutations in patients with dyskeratosis congenita presenting with bone marrow failure and demonstrate that they are deficient in small nucleolar RNA binding. Mice harboring a dyskeratosis congenita germline Npm1 mutation recapitulate both hematological and nonhematological features of dyskeratosis congenita. Thus, our findings indicate that impaired 2′-O-Me can be etiological to human disease.
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Data availability
Raw and preprocessed sequencing and microarray data may be accessed from the Gene Expression Omnibus with accession number GSE135726. Since the informed consent obtained from dyskeratosis congenita patients does not allow for public deposition of the data, the WES data from patient CM108 (NPMD178H) and healthy controls can be communicated upon reasonable request to J.S. and the WES data from the patient harboring the NPM1D180del mutation can be communicated upon reasonable request to I.D. and T.J.V.
Code availability
Code for microarray analysis is available in Supplementary Information 1 file. The code for the WES analysis is available at https://github.com/UCLGeneticsInstitute/DNASeq_pipeline.
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Acknowledgements
D.N. was supported by an EMBO long-term fellowship (no. EMBO-LTF498-2014). K.I. was supported by National Institutes of Health grants (no. R01DK98263, R01DK115577 and R01HL148852), and is a Scholar of The Leukemia and Lymphoma Society. A.H.B. was supported by the Damon Runyon Cancer Research Foundation (no. 2142-12). This work was supported in part by the European Research Council Consolidator (grant no. 311660) and Cancéropole Ile-de-France (no. 2011-1-LABEL-1-AXE2-UP7-3) to J.S; Medical Research Council (grant no. MR/PO18440/1) and Bloodwise (grant no. 14032) to I.D.; the Fondazione AIRC per la Ricerca sul Cancro IG 2016 (grant no. 18568) and the European Research Council Advanced Grant 2016 (no. 740230) to B.F.; and by an Outstanding Investigator Award R35 (grant no. CA197529) and the SHINE grant (no. 5R01DK115536) awarded by National Institutes of Health to P.P.P.
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Contributions
D.N., J.G.C. and P.P.P. designed the experiments and discussed the data. D.N., J.G.C., L.L. and P.P.P. wrote the manuscript. D.N. designed and performed the biochemical and translation-related experiments and analyzed the data. D.N. generated the NPMD180del mouse model, and designed and executed the experiments. S.G. generated the Npm1 conditional knockout mouse model. K.I., S.G. and P.S. performed the conditional Npm1 knockout experiments. A.H.B., A.M. and R.B.D. designed and performed the HiTS-CLIP experiments. D.B. and E.M. performed the computational analysis of the HiTS-CLIP data. Y.Z. designed and performed the northern blot experiments. A.C.B. provided critical reagents. A.G. and C.A.M. provided animal technical assistance. M.P.M. and B.F. provided the human AML samples. K.C. and J.D.L. performed and analyzed the microarray experiments and data. L.L., L.M.M. and O.P. performed the pathology analyses of the hematopoietic features of the NPM1 mutants. T.J.V., I.D., O.B. and J.S. provided and analyzed the dyskeratosis congenita exome dataset. M.S. and S.S. performed and analyzed the PSI-seq experiments and data.
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Supplementary Fig. 1 NPM1 regulates 2’-O-Me through binding of C/D box snoRNAs.
a, Pearson’s Correlation of psi-ratio mean Npm1+/+ and mean Npm1−/− MEFs (n=3 independent experiments). P=0.9. b, NPM1-IP was performed with Npm1+/+ MEFs and snoRNA enrichment was calculated relative to total input. SnordCtrl is snoRD13 that was not identified as an NPM1-bound snoRNA. Data are presented as mean±SD, n=4 independent experiments. c, Western blot analysis of FBL expression levels in Npm1+/+ and Npm1−/− MEFs. HSP90 served as a loading control. Shown is a representative blot out of n=5 independent experiments. d, qPCR analysis of the relative abundance of the specific snoRNAs (x-axis) in Npm1+/+ and Npm1−/− MEFs. Data are presented as mean±SD, n=4 independent experiments.. e, Npm1−/− MEFs were stably transduced to overexpress NPM1 and assessed for specific 2′-O-Me residues (x-axis) levels, mediated by C/D box snoRNAs identified in NPM1 HITS-CLIP. Data are presented as mean±SD of fold change of 2′-O-Me in Npm1−/−+NPM1 MEFs relative to Npm1+/+, n=3 independent experiments. Ctrl residue is the U1804 2′-O-Me modification on the 18S rRNA that is mediated by snoRD20, which was not identified to interact with NPM1 by HITS-CLIP. f, NPM-IP was performed using nuclear extracts, then blotted for NOP58 and DKC1. Neither of the proteins were detected. n=3 independent experiments. g, NPM1-IP was performed using Npm1+/+ MEFs. IP pellets were treated either with RNaseA or MNase for 15min at 37c to digest RNA. FBL was not detected in the treated supernatant (SN) indicating it does not interact with NPM1 via RNA binding. n=3 independent experiments. h, As a positive control, pS6-IP was performed using Npm1+/+ MEFs. pS6-IP pellets were treated either with RNaseA or MNase for 15min at 37c to degrade RNA then blotted for pS6 (for IP validation) and for the RNA binding protein PABP which was released to the supernatant. SN – supernatant. n=3 independent experiments. For all relevant panels, and unless otherwise stated, statistical significance was determined by one-tailed student’s t-test. Uncropped blot images are presented in Supplementary Fig. 11.
Supplementary Fig. 2 NPM1-deletion affects specific modes of translation and does not affect global translation.
a, 35S-Methionine incorporation assay of Npm1+/+ and Npm1−/− MEFs showed no differences on global protein synthesis. Quantity of labeled proteins was analyzed by radiography (RG). n=3 independent experiments. b, HPG metabolic labeling of Npm1+/+ and Npm1−/− MEFs showed no difference in global protein synthesis. Detection of HPG labeling was by azide AlexaFlour-647 and flow cytometry. n=3 independent experiments. c, Flow cytometry analysis of forward scatter and side scatter of Npm1+/+ and Npm1−/− MEFs. n=3 independent experiments. d, Analysis of total RNA content of Npm1+/+ and Npm1−/− MEFs. n=3 independent experiments. e, Northen blot of rRNA processing was performed using Npm1+/+ and Npm1−/− cells. n=3 independent experiments. f, WB analysis of mTOR downstream effectors in Npm1+/+ and Npm1−/− MEFs. HSP90 served as a loading control. n=3 independent experiments. g, Top panel: gene set enrichment (GSEA) analysis using the full set of GFP-positive genes showed statistically significant negative enrichment of putative IRES genes, NES = -1.359, p-value = 1.167e-4. Bottom panel: GSEA analysis of the restricted list of 583 IRES genes identified in Weingarten-Gabbay et al. and found a negative trend of IRES genes associated with Npm1+/- polysome-depleted transcripts, NES = -1.049, p-value = 0.31. h, WB of NPM1 in Npm1+/+, Npm1+/- and Npm1−/− MEFs. Shown is a representative blot of n=3 independent experiments. i, SnoRNA abundance in Npm1+/+ and Npm1+/- MEFs. Data are presented as mean±SD, n=4 independent experiments. j, Levels of specific 2′-O-Me residues (x-axis) mediated by only C/D box snoRNAs identified to interact with NPM1 by HITS-CLIP in Npm1+/- MEFs. Data are presented as mean±SD (n=3 independent experiments) of fold change of 2′-O-Me in Npm1+/- MEFs relative to Npm1+/+. Ctrl residue is the U1804 2′-O-Me modification on the 18S rRNA that is mediated by snoRD20, which was not identified to interact with NPM1 by HITS-CLIP. k, qPCR analysis of Cdkn1b (p27 transcript) and Xiap levels in Npm1−/− MEFs relative to Npm1+/+. Data are presented as mean±SD, n=3 independent experiments. l-m, Ribosome fractionation was performed using Npm1+/+ and Npm1−/− MEFs. ActinB, Gapdh and β2-microglobulin served as cap-translated controls (l). Cdk1nb, Xiap, Vegf and Fgf2 are IRES-translated genes presented in m. mRNA enrichment in polysome fractions is presented as percent from total RNA±SD. n=3 independent experiments. For all relevant panels, and unless otherwise stated, statistical significance was determined by one-tailed student’s t-test. Uncropped blot images are presented in Supplementary Fig. 11.
Supplementary Fig. 3 NPM1-bound snoRNAs modulate translation.
a-d, NIH/3T3 cells were stably transfected with vectors of the PiggyBac transposon system (SBI) to express mouse Snord47 and Snord52, a, Relative snoRNA abundance. Data are presented as mean±SD, n=3 independent experiments. b, Fold increase in 2′-O-Me in snoRNAs overexpressing cells. Data are presented as mean±SD, n=3 independent experiments. c, WB of p27 and XIAP in snoRNA overexpressing cells. RPL22 served as a loading control. Blot is a representative of n=3 independent experiments. d, levels of Cdkn1b and Xiap in snoRNA overexpressing cells. Data are presented as mean±SD, of n=3 independent experiments. Actb served as a control. e-g, Npm1+/+ MEFs were transfected with specific anti-snoRNAs GapmeRs and anti-GFP as control (50nM, Exiqon). e, SnoRNA levels 48hrs after transfection. Data are presented as mean±SD of n=3 independent experiments. f, Levels of the IRES-translated genes XIAP and p27 were reduced due GapmeRs transfection. Blot is a representative of n=3 independent experiments. RPL22 served as a loading control. g, Cdkn1b and Xiap transcripts levels 48 hours after GapmeRs transfection. Data are presented as mean±SD of n=3 independent experiments. h, WB analysis of FLAG expression levels of NPM1, NPMc+ and NPM-RARα in dual luciferase reporter assays. Blot is a representative of n=2 independent experiments. i, Levels of specific 2′-O-Me residues in OCI-AML3 cell line compared to OCI-AML2. Data are presented as mean±SD of n=3 independent experiments. j, SnoRNA abundance in OCI-AML2 (set as 1) and OCI-AML3 cell lines. Data are presented as mean±SD of n=3 independent experiments. For all relevant panels, and unless otherwise stated, statistical significance was determined by one-tailed student’s t-test. Uncropped blot images are presented in Supplementary Fig. 11.
Supplementary Fig. 4 NPM1-depletion and snoRNA-inactivation in K562 cell line.
a, NPM1 KD efficiency in K562/shNPM1 cells compared to scramble transduced cells (Ctrl). αTUBULIN was used as a loading control. Blot is a representative of n=3 independent experiments. b, NPM1-depletion in K562 cells led to decreased 2′-O-Me of specific residues. Data are mean±SD of n=3 independent experiments. Ctrl 2′-O-Me is the 2′-O-Me at 1804 18S rRNA. c, SnoRNA abundance in K562/shNPM1 cells. Data are presented as mean±SD of n=4 independent experiments. d, Examples of indels in specific snoRNA genes generated by CRISPR/Cas9 genome editing in K562 cells. In blue is the specific snoRNA’s wild type sequence; in red are examples of mutations in the same snoRNA induced by CRISPR/Cas9 editing. e, Analysis of control 2′-O-Me 4506 in CRISPR/Cas9 snoRNA-inactivated K562 cells. Data are presented as mean±SD of n=3 independent experiments.. f, Levels of specific 2′-O-Me residues (x-axis) mediated by only C/D box snoRNAs identified to interact with NPM1 by HITS-CLIP. Data are presented as mean±SD, n=3 independent experiments, of fold change of 2′-O-Me in K562/shNPM1+FBL cells relative to K562 cells transduced with scramble vector. Ctrl residue is the U1804 2′-O-Me modification on the 18S rRNA that is mediated by snoRD20, which was not identified to interact with NPM1 by HITS-CLIP. For all relevant panels, and unless otherwise stated, statistical significance was determined by one-tailed student’s t-test.
Supplementary Fig. 5 Characterization of acute Npm1 knock-out in hematopoietic system.
a, Schematic representations of the conditional Npm1 knockout strategy. Map of Hypomorphic allele (top), Npm1 locus after flp-mediated excision of the Neo resistance cassette (2nd) and Npm1 locus after Cre-mediated excision of the floxed exon 1-6 by crossing with an Mx1Cre transgenic mouse (bottom) are shown. Npm1 conditional allele created by insertion of a Frt-flanked Neomycin cassette (NEO) and two loxP sites in appropriate position. The NEO cassette transcriptionally interferes with the Npm1 gene expression, thus giving origin to an intermediate hypomorphic allele. The genomic sequence is depicted as a black line, with black boxes representing exons 1-11. White box represents the neomycin resistance cassette (NEO) and triangles represent the loxP site, respectively. Primers to detect Hypomorphic allele, NEO-free allele and Excised allele were also shown. b, PCR analysis of genomic DNA from the Linneg cells of NpmF/FMx1Cre+ or NpmF/FMx1Cre- mice after pIpC injection with the primers Fw3 and Rev3. The positions of amplified fragment corresponding to excised allele are indicated. n=3 independent experiments. c-d, Reduction of Npm1 after pIpC injection is confirmed at mRNA level (c) and protein level (d). Residual Npm1 expression in MNCs and CD34negKSL cells (c) and levels of NPM1 in Linpos and Linneg cells (d) are analyzed 14 days after pIpC injection. c, Data are presented as mean±SD of n=6 independent experiments. d, n=3 independent experiments. For all relevant panels, and unless otherwise stated, statistical significance was determined by one-tailed student’s t-test.
Supplementary Fig. 6 Npm1-deletion leads to bone marrow failure in adult mice.
a, Peripheral blood counts of Npm1F/FMx1Cre- and Npm1F/FMx1Cre+ mice 4 weeks after pIpC injections, demonstrating low WBC and low platelets. Data are presented as mean±SD of n=4 biologically independent animals. *P=0.019, **P=0.003. b, Npm1-deletion in adult LSK cells leads to impairment of maintenance of quiescence. % of cells in G0 phase in Npm1-deleted LSK cells 4 days after pIpC injection. Data are presented as mean±SD of n=4 biologically independent animals. c, Purified LSK cells from Npm1F/F mice were transduced with MSCV Puro-IRES-GFP (Empty) or MSCV Puro-Cre-IRES-GFP (Cre). Single sorted GFP+ cells were cultured with SCF+TPO and cell division was monitored daily. First division day was determined in 100 cells per experiment. Data are presented as mean±SD of n=3 biologically independent animals. d, Sorted LSK cells from NpmF/FMx1Cre+ or Mx1Cre- LSK were transplanted with competitor BMMNCs. 8 weeks after BMT, pIpC was injected into the recipient mice, and %G0 phase in donor-derived (Ly45.2) LSK cells was determined by PyroninY staining. Data are presented as mean±SD of n=3 biologically independent animals. e, Colony forming capacity (left) and high proliferation potential (HPP) colony forming cells (right) were determined. Data are presented as mean±SD of n=4 biologically independent animals. f, Npm1-deletion leads to significant reduction of long-term culture-initiating cells (LTC-IC). LSK cells sorted from pIpC treated NpmF/FMx1Cre- or NpmF/FMx1Cre+ or Npm+/+Mx1Cre- or Npm+/+Mx1Cre+ mice were cultured with stromal cells for 6 weeks, and tested for colony formation. Frequencies of LTC-IC in limiting-dilution assay were determined using Poisson statistics. n=4 biologically independent animals. g, Survival of Npm1/Trp53 compound mutant (n=16 biologically independent animals per group). h, Npm1/Trp53 double-null displays myeloproliferation. Two weeks after pIpC injection, myeloid compartment in bone marrow was measured by CD11b/Gr-1 positivity. Data are presented as mean±SD of n=3 biologically independent animals per group. i, Two weeks after pIpC injection, B220, CD3 and CD11b and/or Gr-1 (Myeloid) positive cells in spleen were assessed. For all relevant panels, and unless otherwise stated, statistical significance was determined by one-tailed student’s t-test.
Supplementary Fig. 7 NPMD178H is deficient in snoRNA binding.
a, Npm1−/− MEFs were stably transduced either with NPM1D178H-FLAG, or NPM1D178H-FLAG or NPM1wt-FLAG (set as 1, not shown). FLAG-IP was performed and Snord enrichment was analyzed by qPCR. Fold enrichment was calculated relative to the enrichment in NPM1wt-FLAG IP. Data are presented as mean±SD of n=3 independent experiments, b, NPM1 mutants associate as wild type NPM with FBL. Npm1−/− MEFs were stably transduced either with NPM1D178H-FLAG, NPM1D180del-FLAG or NPM1wt-FLAG. FLAG-IP was performed and co-precipitation with FBL was assessed through WB. Representative blot of n=3 independent experiments. c, snoRNA levels in BV311 and CM108 cells. Data are presented as mean±SD of n=3 independent experiments. SNORD13 served as a control (SNORDCtrl). d, WB analysis of CM108 and BV311 cells. n=2 independent experiments e, qPCR analysis of CDKN1B and XIAP levels in CM108 patient cells compared to BV311 control cells. ACTB served as a non-IRES control. Data are presented as mean±SD of n=3 independent experiments. f, 35S-Methionine incorporation assay to test for global translation comparing BV311 and CM108 cells. Shown is a representative blot of n=3 independent experiments. g, WB analysis of mTOR downstream effectors in BV311 (control) and CM108 (DC patient) cells. GAPDH served as a loading control. Shown is a representative blot of n=3 independent experiments. h, Northen blot of rRNA processing was performed using BV311 and CM108 cells. Shown is a representative blot of n=2 independent experiments. i, Traces of polysome fractionation of BV311 and CM108 cells. The traces are shown offset from one another on the arbitrary y axis (absorbance at 254 nm) for ease of visualizing the data. Shown is a representative trace of n=3 independent experiments. j, NPM1 localizes to the nucleolus in CM108 patient samples. Immunofluorescence (IF) was performed with anti-NPM1 (red), anti-FBL (as a nucleolus marker, green) and DAPI (blue). Shown are representative pictures of n=3 independent experiments. Scale bar–10μm. k, Similar levels of p53 and p21 were observed in patient cells (CM108) and control (BV311). HSP90 served as loading control. Shown is a representative blot of n=2 independent experiments. l, IF of censtrosomes (γ-tubulin) showed similar numbers in patient (CM108) and control (BV311) cells. Data are presented as mean±SD of n=5 (BV311) and n=9 (CM108) independent experiments. For all relevant panels, and unless otherwise stated, statistical significance was determined by one-tailed student’s t-test. Uncropped blot images are presented in Supplementary Fig. 11.
Supplementary Fig. 8 NPM1D180del is deficient in snoRNA binding.
a, Traces of polysome fractionation of NPM1D180del CRISPR MEFs (orange) and Trp53−/− parental MEFs (black). Shown are representative traces of n=3 independent experiments. b, Firefly/Renilla activity ratio in NPM1D180del CRISPR MEFs was calculated relative to the ratio measured in Trp53−/− parental MEFs. Data are presented as mean±SD of n=5 independent experiments. c, Western blot analysis of the levels of NPM and of the IRES translated genes XIAP and p27 in NPM1D180del CRISPR MEFs. TUBULIN served as a loading control. Blot is a representative of n=2 independent experiments. d, FBL-IP was performed using NPM1D180del CRISPR MEFs and parental Trp53−/− MEFs as control. SnoRNA enrichment was calculated relative to total input and fold enrichment in control MEFs. SnordCtrl is Snord13. Data are presented as mean±SD of n=3 independent experiments. e, Analysis of specific 2′-O-Me (x-axis) in NPM1D180del CRISPR MEFs compared to control MEFs. Data are presented as mean±SD of n=3 independent experiments. Ctrl 2′-O-Me is the 2′-O-Me at 1804 18S rRNA. f, WB validation of FLAG expression in K562/shNPM1 cells. RPL22 served as a loading control. Shown is a representative blot of n=2 independent experiments. For all relevant panels, and unless otherwise stated, statistical significance was determined by one-tailed student’s t-test. Uncropped blot images are presented in Supplementary Fig. 11.
Supplementary Fig. 9 NPM1D180del mice display features of aberrant hematopoiesis.
a, HSC compartment analysis of 2 months old NPM1D180del mice (Heterozygous, n=4, Homozygous, n=3) and control littermates (n=6). Data are presented as mean±SD b, Progenitors analysis of 2 months old NPM1D180del mice (Heterozygous, n=3, Homozygous, n=2) and control littermates (n=4). Data are presented as mean±SD c, Analysis of erythroblast percentage in the bone marrow of 2 months old NPM1D180del mice (Heterozygous, n=4, homozygous, n=3) compared to littermates mice (n=7). Data are presented as mean±SD. d, Red blood counts (RBC) and platelet counts (PLT) of heterozygous (orange circles, n=5) and homozygous (red circles, n=5) NPM1D180del mice compared to control littermates (n=12). Data are presented as mean±SD. e, Representative peripheral blood smears (out of n=9 independent samples) of NPM1D180del mice at six months of age show dysplastic RBC cells (i-control, ii-pencil cells (green arrows); iii-target cells (black arrows)), and dysplastic neutrophils (iv-control, v-vi-dysplastic cells, red arrow heads). Scale bar −10µm. f-i, An NPM1D180del mouse developed myeloid proliferative disorder exhibited splenomegaly (f) and pail bones (g). h, FACS analysis of the BM of the NPM1D180del mouse showed very low frequency of erythrocytes (TER119+ cells) and significant high percentage of myeloid cells (Gr1+CD11b+ cells), compared to littermate control. i, FACS analysis of the spleen of the NPM1D180del mouse demonstrated high percentage of TER119+ cells and of myeloid cells (Gr1+CD11b+ cells), compared to littermate control. j, western blot analysis of the IRES translated genes p27 and XIAP, in total BM cells of NPM1D180del and control littermates, demonstrating reduced protein levels in NPM1D180del cells. n=3 independent experiments performed. k, snoRNAs are expressed in similar levels in NPM1D180del mice and control littermates. Data are presented as mean±SD of n=3 independent experiments. For all relevant panels, and unless otherwise stated, statistical significance was determined by one-tailed student’s t-test. Uncropped blot images are presented in Supplementary Fig. 11.
Supplementary Fig. 10 Model of NPM1 function as a 2′-O-Me modulator.
a, NPM1 proteins assemble in pentamers and bind snoRNAs as well as FBL to facilitate snoRNA loading, proper rRNA 2′-O-methylation and normal hematopoiesis. b, NPM mutants identified in DC patients affect the complex’s ability to bind snoRNAs and hence snoRNA loading into snoRNPs is compromised. This leads to low 2′-O-Me, translational deficiencies in HSCs and to aberrant hematopoiesis. c, TCGA database reveals additional mutations in the specific NPM1 acidic repeat identified in various solid tumors.
Supplementary Fig. 11
Uncropped scans of western blot data.
Supplementary information
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Supplementary Figs. 1–11
Supplementary Tables
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Supplementary Software
Microarray analysis code
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Nachmani, D., Bothmer, A.H., Grisendi, S. et al. Germline NPM1 mutations lead to altered rRNA 2′-O-methylation and cause dyskeratosis congenita. Nat Genet 51, 1518–1529 (2019). https://doi.org/10.1038/s41588-019-0502-z
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DOI: https://doi.org/10.1038/s41588-019-0502-z
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