Ribosomal protein RPL11 haploinsufficiency causes anemia in mice via activation of the RP-MDM2-p53 pathway

Abstract

Recent discovery of the ribosomal protein (RP) RPL11 interacting with and inhibiting the E3 ubiquitin ligase function of MDM2 established the RP-MDM2-p53 signaling pathway, which is linked to biological events, including ribosomal biogenesis, nutrient availability, and metabolic homeostasis. Mutations in RPs lead to a diverse array of phenotypes known as ribosomopathies in which the role of p53 is implicated. Here, we generated conditional RPL11-deletion mice to investigate in vivo effects of impaired RP expression and its functional connection with p53. While deletion of one Rpl11 allele in germ cells results in embryonic lethality, deletion of one Rpl11 allele in adult mice does not affect viability but leads to acute anemia. Mechanistically, we found RPL11 haploinsufficiency activates p53 in hematopoietic tissues and impedes erythroid precursor differentiation, resulting in insufficient red blood cell development. We demonstrated that reducing p53 dosage by deleting one p53 allele rescues RPL11 haploinsufficiency-induced inhibition of erythropoietic precursor differentiation and restores normal red blood cell levels in mice. Furthermore, blocking the RP-MDM2-p53 pathway by introducing an RP-binding mutation in MDM2 prevents RPL11 haploinsufficiency–caused p53 activation and rescues the anemia in mice. Together, these findings demonstrate that the RP-MDM2-p53 pathway is a critical checkpoint for RP homeostasis and that p53-dependent cell cycle arrest of erythroid precursors is the molecular basis for the anemia phenotype commonly associated with RP deficiency. Recent discovery of the ribosomal protein (RP) RPL11 interacting with and inhibiting the E3 ubiquitin ligase function of MDM2 established the RP-MDM2-p53 signaling pathway, which is linked to biological events, including ribosomal biogenesis, nutrient availability, and metabolic homeostasis. Mutations in RPs lead to a diverse array of phenotypes known as ribosomopathies in which the role of p53 is implicated. Here, we generated conditional RPL11-deletion mice to investigate in vivo effects of impaired RP expression and its functional connection with p53. While deletion of one Rpl11 allele in germ cells results in embryonic lethality, deletion of one Rpl11 allele in adult mice does not affect viability but leads to acute anemia. Mechanistically, we found RPL11 haploinsufficiency activates p53 in hematopoietic tissues and impedes erythroid precursor differentiation, resulting in insufficient red blood cell development. We demonstrated that reducing p53 dosage by deleting one p53 allele rescues RPL11 haploinsufficiency-induced inhibition of erythropoietic precursor differentiation and restores normal red blood cell levels in mice. Furthermore, blocking the RP-MDM2-p53 pathway by introducing an RP-binding mutation in MDM2 prevents RPL11 haploinsufficiency–caused p53 activation and rescues the anemia in mice. Together, these findings demonstrate that the RP-MDM2-p53 pathway is a critical checkpoint for RP homeostasis and that p53-dependent cell cycle arrest of erythroid precursors is the molecular basis for the anemia phenotype commonly associated with RP deficiency. The tumor suppressor p53 is the most commonly mutated gene across many types of cancers (1Hollstein M. Sidransky D. Vogelstein B. Harris C.C. p53 mutations in human cancers.Science. 1991; 253: 49-53Crossref PubMed Scopus (7501) Google Scholar, 2Olivier M. Hollstein M. Hainaut P. TP53 mutations in human cancers: origins, consequences, and clinical use.Cold Spring Harbor Perspect. Biol. 2010; 2: a001008Crossref PubMed Scopus (1264) Google Scholar). p53-mediated cell cycle arrest and apoptosis in response to DNA damage and other cellular stresses are thought to be vital for p53-mediated tumor suppression, a notion that led to p53 being nicknamed the “Guardian of the Genome” (3Lane D.P. p53, guardian of the genome.Nature. 1992; 358: 15-16Crossref PubMed Scopus (4559) Google Scholar). p53 is tightly regulated by the E3 ubiquitin ligase MDM2 via direct binding and increased proteosomal degradation via MDM2-mediated p53 polyubiquitination (4Moll U.M. Petrenko O. The MDM2-p53 interaction.Mol. Cancer Res. 2003; 1: 1001-1008PubMed Google Scholar). The initial observation that ribosomal protein (RP) RPL11 binds to and inhibits the E3 ligase function of MDM2 led to the discovery of the RP-MDM2-p53 pathway, which involves multiple RPs and has been shown to contribute to tumor suppression, nutrient balance, and metabolic homeostasis (5Zhang Y. Lu H. Signaling to p53: ribosomal proteins find their way.Cancer Cell. 2009; 16: 369-377Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 6Liu Y. Deisenroth C. Zhang Y. RP-MDM2-p53 pathway: linking ribosomal biogenesis and tumor surveillance.Trends Cancer. 2016; 2: 191-204Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Further studies demonstrated that p53 is activated in response to imbalances in RP stoichiometry caused by oncogene activation or RNA interference (7Grandori C. Gomez-Roman N. Felton-Edkins Z.A. Ngouenet C. Galloway D.A. Eisenman R.N. et al.c-Myc binds to human ribosomal DNA and stimulates transcription of rRNA genes by RNA polymerase I.Nat. Cell Biol. 2005; 7: 311-318Crossref PubMed Scopus (521) Google Scholar, 8Nicolas E. Parisot P. Pinto-Monteiro C. de Walque R. De Vleeschouwer C. Lafontaine D.L. Involvement of human ribosomal proteins in nucleolar structure and p53-dependent nucleolar stress.Nat. Commun. 2016; 711390Crossref Scopus (124) Google Scholar). These studies established a link between p53 and ribosomal biogenesis, a vital and energetically demanding process in all eukaryotic cells (9Warner J.R. The economics of ribosome biosynthesis in yeast.Trends Biochem. Sci. 1999; 24: 437-440Abstract Full Text Full Text PDF PubMed Scopus (1455) Google Scholar). Mutations in RPs lead to a class of diseases known as ribosomopathies with wide ranging phenotypes (10Narla A. Ebert B.L. Ribosomopathies: human disorders of ribosome dysfunction.Blood. 2010; 115: 3196-3205Crossref PubMed Scopus (595) Google Scholar), in which the role of p53 has been implicated (11Fumagalli S. Thomas G. The role of p53 in ribosomopathies.Semin. Hematol. 2011; 48: 97-105Crossref PubMed Scopus (74) Google Scholar). However, the mechanism underlying RP mutation-dependent p53 activation and subsequent ribosomopathy is not fully understood. RPL11 was the first RP identified to interact with and inhibit the E3 ligase function of MDM2, leading to p53 stabilization and activation (12Lohrum M.A. Ludwig R.L. Kubbutat M.H. Hanlon M. Vousden K.H. Regulation of HDM2 activity by the ribosomal protein L11.Cancer Cell. 2003; 3: 577-587Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar, 13Zhang Y. Wolf G.W. Bhat K. Jin A. Allio T. Burkhart W.A. et al.Ribosomal protein L11 negatively regulates oncoprotein MDM2 and mediates a p53-dependent ribosomal-stress checkpoint pathway.Mol. Cell. Biol. 2003; 23: 8902-8912Crossref PubMed Scopus (461) Google Scholar). Of the multiple MDM2-binding RPs, RPL11 appears to be particularly important for p53 regulation. RPL11 is protected from proteasomal degradation upon ribosomal biogenesis stress, while many other MDM2-binding RPs, including RPL23, RPL25, and RPS7, are degraded upon ribosomal stress (14Bursac S. Brdovcak M.C. Pfannkuchen M. Orsolic I. Golomb L. Zhu Y. et al.Mutual protection of ribosomal proteins L5 and L11 from degradation is essential for p53 activation upon ribosomal biogenesis stress.Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 20467-20472Crossref PubMed Scopus (157) Google Scholar). RPS6 deletion impairs ribosome biogenesis and specifically induces expression of RPL11, but not other RPs, to cause p53 activation (15Fumagalli S. Di Cara A. Neb-Gulati A. Natt F. Schwemberger S. Hall J. et al.Absence of nucleolar disruption after impairment of 40S ribosome biogenesis reveals an rpL11-translation-dependent mechanism of p53 induction.Nat. Cell Biol. 2009; 11: 501-508Crossref PubMed Scopus (266) Google Scholar). Upon ribosomal stress, RPL11 interacts with p14ARF (p19Arf in mouse) to augment p53 activation (16Dai M.S. Challagundla K.B. Sun X.X. Palam L.R. Zeng S.X. Wek R.C. et al.Physical and functional interaction between ribosomal protein L11 and the tumor suppressor ARF.J. Biol. Chem. 2012; 287: 17120-17129Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar). RPL11, but not other RPs, promotes MDM2-mediated MDMX polyubiquitination and degradation (17Gilkes D.M. Chen L. Chen J. MDMX regulation of p53 response to ribosomal stress.EMBO J. 2006; 25: 5614-5625Crossref PubMed Scopus (113) Google Scholar). Recent studies have shown that upon c-MYC overexpression, the p19Arf-MDM2-p53 oncogenic insult response pathway and the RPL11-MDM2-p53 ribosomal stress response pathway are two nonredundant mechanisms possessing similar capabilities for p53 activation, indicating that, like p19Arf, RPL11 acts as a tumor suppressor (18Meng X. Carlson N.R. Dong J. Zhang Y. Oncogenic c-Myc-induced lymphomagenesis is inhibited non-redundantly by the p19Arf-Mdm2-p53 and RP-Mdm2-p53 pathways.Oncogene. 2015; 34: 5709-5717Crossref PubMed Scopus (21) Google Scholar). To date, at least 220 distinct mutations in 18 RP genes are reported in Diamond-Blackfan anemia (DBA) patients, and about 50% of all DBA patients can be characterized by mutations in four RPs, the RPL11, RPL5, RPS19, and RPL26 (19Horos R. Ijspeert H. Pospisilova D. Sendtner R. Andrieu-Soler C. Taskesen E. et al.Ribosomal deficiencies in Diamond-Blackfan anemia impair translation of transcripts essential for differentiation of murine and human erythroblasts.Blood. 2012; 119: 262-272Crossref PubMed Scopus (129) Google Scholar). Interestingly, RPS19 and RPS26 activate p53 in an RPL11-dependent manner (20Dutta S. Akey I.V. Dingwall C. Hartman K.L. Laue T. Nolte R.T. et al.The crystal structure of nucleoplasmin-core: implications for histone binding and nucleosome assembly.Mol. Cell. 2001; 8: 841-853Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 21Cui D. Li L. Lou H. Sun H. Ngai S.M. Shao G. et al.The ribosomal protein S26 regulates p53 activity in response to DNA damage.Oncogene. 2014; 33: 2225-2235Crossref PubMed Scopus (78) Google Scholar), underscoring a role for RPL11 in linking DBA with the function of p53. Studies in zebrafish have shown that p53 activation is responsible for the morphological deficiencies associated with RPL11 depletion (22Chakraborty A. Uechi T. Nakajima Y. Gazda H.T. O'Donohue M.F. Gleizes P.E. et al.Cross talk between TP53 and c-Myc in the pathophysiology of Diamond-Blackfan anemia: evidence from RPL11-deficient in vivo and in vitro models.Biochem. Biophys Res. Commun. 2018; 495: 1839-1845Crossref PubMed Scopus (11) Google Scholar). In contrast to the zebrafish study, however, a study in mice indicated that RPL11 deletion decreases p53 activity (23Morgado-Palacin L. Varetti G. Llanos S. Gomez-Lopez G. Martinez D. Serrano M. Partial loss of Rpl11 in adult mice recapitulates Diamond-Blackfan anemia and promotes lymphomagenesis.Cell Rep. 2015; 13: 712-722Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). the is to is To we generated RPL11 deletion mice. study the phenotype in RPL11 deletion however, to the study of p53 study an of p53 function upon RPL11 deletion in mice. To the in vivo function of RPL11, we generated mice a conditional Rpl11 was by two We RPL11 mice with which the to one allele of mice from generated from the indicating that Rpl11 is embryonic is with a a gene that Rpl11 is insufficient to embryonic (23Morgado-Palacin L. Varetti G. Llanos S. Gomez-Lopez G. Martinez D. Serrano M. Partial loss of Rpl11 in adult mice recapitulates Diamond-Blackfan anemia and promotes lymphomagenesis.Cell Rep. 2015; 13: 712-722Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). To the embryonic caused by Rpl11 we mice with which a to mice and the mice to mice. We mice with to Rpl11 mice after indicating that deletion of RPL11 in adult mice embryonic cells after with to RPL11 In RPL11 deletion as and for the cells with RPL11 deletion and of RPL11 expression of other RPs in tissues and in cells but their expression indicating the is RPL11 deletion protein by The of protein was to a of as loss of RPL11 of ribosomal and of ribosome and In loss of RPL11 in adult mice inhibits protein of cell and the other RPL11 deletion generated by mice with not cause cause and mice the and of multiple including and as with We that of the from mice and of anemia. of blood a of after was In was increased in mice in is a of in response to the acute loss of R. Scholar). RPL11 haploinsufficiency in the which blood and blood in differentiation as demonstrated by in and in to the mice as shown by to mice of the structure was in the with a in that contribute to the increased Together, these demonstrated that deletion of one Rpl11 allele in mice of anemia and To the RPL11 haploinsufficiency-induced we for erythroid precursor cell differentiation by of cells the cell and of and and and of and in the that impaired erythroid cell M. R. E. J.R. D. M. and of erythroid the 2011; Scholar, M. H. C. in mice to of erythroblasts.Blood. 2001; PubMed Scopus Google Scholar). The impaired erythroid in the was demonstrated by of erythroid cells To mechanisms underlying the of we cell cycle for erythroid precursor of and of in the and cell in the to and indicating a cell cycle arrest in the erythroid The cells and the which are not in cell cycle These that loss of RPL11 induces erythroid cell cycle arrest that To the in mice was to hematopoietic we We mice with to one Rpl11 allele We mice with to as after the or cells and mice and to the mice and levels as as after while their indicating an of the To RPL11 hematopoietic cell and we by and cells at a and the mice. Interestingly, mice normal of and indicating that the cells in the cell The of cells was demonstrated by blood the erythroid cell of mice was not in the mice these indicated that RPL11 haploinsufficiency the of the cells and that the anemia in mice is by cell of erythroid precursor studies that RPL11 in zebrafish leads to p53 activation (22Chakraborty A. Uechi T. Nakajima Y. Gazda H.T. O'Donohue M.F. Gleizes P.E. et al.Cross talk between TP53 and c-Myc in the pathophysiology of Diamond-Blackfan anemia: evidence from RPL11-deficient in vivo and in vitro models.Biochem. Biophys Res. Commun. 2018; 495: 1839-1845Crossref PubMed Scopus (11) Google Scholar). However, a study in mice that RPL11 p53 (23Morgado-Palacin L. Varetti G. Llanos S. Gomez-Lopez G. Martinez D. Serrano M. Partial loss of Rpl11 in adult mice recapitulates Diamond-Blackfan anemia and promotes lymphomagenesis.Cell Rep. 2015; 13: 712-722Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). To the of RPL11 p53 we and mice with and p53 expression in tissues after the to the of anemia. We found that the protein levels of p53 increased in the and increased in the and or in other tissues of the levels increased in the and of the indicating that RPL11 deletion activates p53 in tissues of The hematopoietic p53 activation was by of was in and tissues of mice To the hematopoietic p53 activation in mice was cell we p53 in mice. In the and tissues of p53 and which was not in tissues of mice the RPL11 haploinsufficiency–caused p53 activation was cell To p53 activation is responsible for the phenotype in the RPL11 haploinsufficiency we generated of and mice. These mice with and their blood RPL11 deletion of a p53 allele and and RPL11 deletion of one p53 allele and and of one p53 allele levels and of the mice deletion of one p53 allele RPL11 of p53 RPL11 haploinsufficiency–caused however, we not these deletion of p53 results in tumor at in mice The a for and Cancer Biol. 7: PubMed Scopus Google Scholar), the results To the effects of p53 deletion we the cell cycle of the erythroid precursor in the In contrast to cell cycle arrest found in the cell cycle from similar to that of the and that p53 a critical role in the of erythropoietic disorders by cell cycle arrest in erythroid precursor To the hematopoietic in the mice that is to p53 activation, we and tissues from mice to p53 expression with p53 and MDM2 by p53 deletion of RPL11 in p53 stabilization and activation and which was by deletion of a p53 allele and Together, these demonstrated that the phenotype in the mice is by deletion of a p53 that p53 activation is the of these Recent studies an RP-MDM2-p53 stress response pathway by RP binding to and inhibiting the E3 ligase function of MDM2 (5Zhang Y. Lu H. Signaling to p53: ribosomal proteins find their way.Cancer Cell. 2009; 16: 369-377Abstract Full Text Full Text PDF PubMed Scopus (463) Google Scholar, 6Liu Y. Deisenroth C. Zhang Y. RP-MDM2-p53 pathway: linking ribosomal biogenesis and tumor surveillance.Trends Cancer. 2016; 2: 191-204Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). a mutation in the of MDM2 which inhibits the binding between MDM2 and RPL11 M.S. Jin A. Deisenroth C. Wolf G. Zhang Y. mutations in the MDM2 ribosomal protein interaction and p53 Cell. Biol. PubMed Scopus Google Scholar), p53 response to ribosomal stress E. Jin A. Deisenroth C. Bhat K. H. M.S. et tumor pathway by ribosomal Cell. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. Y. Jin A. L. et al.Ribosomal pathway nutrient stress with by and Natl. Acad. Sci. U. S. A. 2014; PubMed Scopus Google Scholar). To the RP-MDM2-p53 pathway is in RPL11 haploinsufficiency caused we mice with mice to mice. We the mice with to mice. The of mutation anemia and indicating that an RP-MDM2-p53 pathway is for hematopoietic inhibition in response to RPL11 the RPL11 p53 activation was by the mutation at protein and and RPL11 haploinsufficiency not RP expression levels but MDM2 binding to RPL5, RPL23, and RPL11 we that RPL11 haploinsufficiency augment MDM2 binding with and RPL11 to which was been by mutation to these demonstrated that RPL11 haploinsufficiency activates p53 by MDM2 binding to studies have indicated between p53 activation and ribosomopathies as the p53 of however, P. S. K. Zhang Y. J. M.S. et of the interaction the erythroid in a for Diamond-Blackfan 2015; PubMed Scopus Google Scholar, H. M. T. C. A. Y. et hematopoietic cells from DBA patients with mutations in RPL11 and RPS19 genes distinct erythroid phenotype in 2012; 3: PubMed Scopus Google Scholar). that RPL11 hematopoietic is p53 but increased p53 activity is not to in mice. studies that mice with a of p53, which increased p53 have phenotype and normal blood and levels S. J. S. N. H. et mice that PubMed Scopus Google Scholar). the which an p53 a increased p53 DNA damage and to tumor evidence of I. M. J. P. et mice DNA damage are tumor and J. PubMed Scopus Google Scholar). that p53 activation is not to cause and other p53 activity are in and have shown that of RP genes of ribosomopathy and The basis by which RP mutations understood. signaling in cell stress is diseases caused by RP deficiency. in RPs has been shown to p53 which multiple ribosomopathies are associated with We that activation of p53 in the hematopoietic cells leads to of these cells or for p53 mutation to cell of p53 cell cycle and metabolic is which of these is important for RP indicated that RPL11 haploinsufficiency cell cycle arrest specifically in in erythroid which is the cause RP anemia. cells RP to of ribosome a regulation of RP is across multiple levels of protein including and protein degradation of ribosomal PubMed Scopus Google Scholar). that of ribosomal biosynthesis and activation of p53 RP mutation are two for the of Interestingly, deletion of one Rpl11 allele not cause of RPL11 and other RP protein activated p53 We that in the cells RPL11 is by transcription and translation of the Rpl11 allele or by RPL11 of RPL11 levels ribosomal biogenesis, resulting in increased and p53 The many of the of a RPL11 deletion study (23Morgado-Palacin L. Varetti G. Llanos S. Gomez-Lopez G. Martinez D. Serrano M. Partial loss of Rpl11 in adult mice recapitulates Diamond-Blackfan anemia and promotes lymphomagenesis.Cell Rep. 2015; 13: 712-722Abstract Full Text Full Text PDF PubMed Scopus Google Scholar). However, the study indicated that p53 activity is in the leading to increased lymphomagenesis in the results that in the of cellular stress, p53 activity is increased in the mice We not have about the To the increased p53 activity is responsible for RPL11 we a p53 allele in the mice to mice. of one p53 allele all the phenotypes of mice. of the deletion mice established that a dosage of p53 is for RPL11 that p53 activation is the cause of the anemia of the mice. and the mutation a mutation in MDM2 RP we that an increased RP binding to MDM2 is for p53 activation and anemia phenotype in the mice. The study demonstrated that RPL11 deletion a ribosomal stress that is to p53 RP binding to MDM2, a role for the RP-MDM2-p53 pathway in RP stress to p53. We that the of RPL11 anemia by mutation appears to be of levels in the mice and of the We that is of the mutation binding of but all RPs and RPs that to RPL11 deletion caused ribosomal stress to activate p53. studies have shown that mutation and RPL11 binding but not binding M.S. Jin A. Deisenroth C. Wolf G. Zhang Y. mutations in the MDM2 ribosomal protein interaction and p53 Cell. Biol. PubMed Scopus Google Scholar, E. Jin A. Deisenroth C. Bhat K. H. M.S. et tumor pathway by ribosomal Cell. 2010; Full Text Full Text PDF PubMed Scopus Google Scholar). that as many as RPs have been identified to MDM2 and p53 activity Zhang pathway: linking ribosomal biogenesis and tumor surveillance.Trends Cancer. 2016; 2: 191-204Abstract Full Text Full Text PDF PubMed Scopus Google Scholar), is that of these RPs that to inhibit function to activate p53. The activation of p53 hematopoietic tissues in the mice is particularly and phenotype is not to RPL11 deletion as of and genes induces similar p53 activation the erythroid S. A. K. A. N. C. et for ribosomal protein genes activation of p53 in human erythroid 2011; PubMed Scopus Google Scholar). The mechanism of cell p53 activation is but we that the of erythroid precursors to which a that is particularly to RP Furthermore, studies have shown that and as erythroid ribosome that stresses the translation erythroid cells J. R. regulation of a ribosome pathway in erythroid cells and Rep. 2016; Full Text Full Text PDF PubMed Scopus Google Scholar). erythroid precursors are to p53 activation in response to RP expression and and mice with or and a and cycle with from to mice and of and mice for the The and at the of all studies was in a of at to with The and was at for with or other for a of in the are from the initial was from the or the via to to the at the of which is in by an to the Cancer the to blood of blood was upon a being the of a of the for to and with an with a and mice with as to for and of mice to the with or or a of and from the cells and with and for at with The cells a are shown as a of of the cell cycle cells and with and for at the cells and by for cells and by with and by for to erythroid the and The DNA of these by the for cell cycle an RNA was from tissues RNA was with a and was by was was with the as the in in a with and in with and activation of in was to the and to from to RPL11 and E. 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