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Ribosomal protein S19 and S24 insufficiency cause distinct cell cycle defects in Diamond-Blackfan anemia
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology.ORCID iD: 0000-0002-4383-9880
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2009 (English)In: Biochimica et Biophysica Acta, ISSN 0006-3002, E-ISSN 1878-2434, Vol. 1792, no 10, 1036-1042 p.Article in journal (Refereed) Published
Abstract [en]

Diamond-Blackfan anemia (DBA) is a severe congenital anemia characterized by a specific decrease of erythroid precursors. The disease is also associated with growth retardation, congenital malformations, a predisposition for malignant disease and heterozygous mutations in either of the ribosomal protein (RP) genes RPS7, RPS17, RPS19, RPS24, RPL5, RPL11 and RPL35a. We show herein that primary fibroblasts from DBA patients with truncating mutations in RPS19 or in RPS24 have a marked reduction in proliferative capacity. Mutant fibroblasts are associated with extended cell cycles and normal levels of p53 when compared to w.t. cells. RPS19 mutant fibroblasts accumulate in the G1 phase, whereas the RPS24 mutant cells show an altered progression in the S phase resulting in reduced levels in the G2/M phase. RPS19 deficient cells exhibit reduced levels of Cyclin-E, CDK2 and retinoblastoma (Rb) protein supporting a cell cycle arrest in the G1 phase. In contrast, RPS24 deficient cells show increased levels of the cell cycle inhibitor p21 and a seemingly opposing increase in Cyclin-E, CDK4 and CDK6. In combination, our results show that RPS19 and RPS24 insufficient fibroblasts have an impaired growth caused by distinct blockages in the cell cycle. We suggest this proliferative constraint to be an important contributing mechanism for the complex extra-hematological features observed in DBA.

Place, publisher, year, edition, pages
2009. Vol. 1792, no 10, 1036-1042 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-110062DOI: 10.1016/j.bbadis.2009.08.002ISI: 000271071600012PubMedID: 19689926OAI: oai:DiVA.org:uu-110062DiVA: diva2:274890
Available from: 2009-11-02 Created: 2009-11-02 Last updated: 2017-12-12Bibliographically approved
In thesis
1. Ribosomal Proteins in Diamond-Blackfan Anemia: Insights into Failure of Ribosome Function
Open this publication in new window or tab >>Ribosomal Proteins in Diamond-Blackfan Anemia: Insights into Failure of Ribosome Function
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Diamond-Blackfan anemia (DBA) is a severe congenital anemia characterized by a defect in red blood cell production. The disease is associated with growth retardation, malformations, a predisposition for malignant disease and heterozygous mutations in either of the ribosomal protein (RP) genes RPS7, RPS17, RPS19, RPS24, RPL5, RPL11 and RPL35a.

In a cellular model for DBA, siRNA knock-down of RPS19 results in a relative decrease of other ribosomal (r) proteins belonging to the small subunit (RPS20, RPS21, RPS24) when compared to r-proteins from the large ribosomal subunit (RPL3, RPL9, RPL30, RPL38). RPS19 mutant cells from DBA patients show a similar and coordinated down-regulation of small subunit proteins. The mRNA levels of the small subunit r-proteins remain relatively unchanged. We also show that RPS19 has an extensive number of transcriptional start sites resulting in mRNAs of variable 5’UTR length. The short variants are translated more efficiently. Structural sequence variations in the 5’UTR of RPS19 found in DBA patients show a 20%-30% reduced translational activity when compared to normal transcripts.

Primary fibroblast from DBA patients with truncating mutations in RPS19 or RPS24 showed specific cell cycle defects. RPS19 mutant fibroblasts accumulate in the G1 phase whereas the RPS24 mutant cells show a defect in G2/M phase. The G1 phase arrest is associated with a reduced level of phosphorylated retinoblastoma (Rb) protein, cyclin E and cdk2 whereas the G2/M phase defect is associated with increased levels of p21, cyclin E, cdk4 and cdk6.

RPS19 interacts with PIM-1 kinase. We investigated the effects of targeted disruptions of both Rps19 and Pim-1 in mice. Double mutant (Rps19+/-, Pim-1-/-) mice have increased peripheral white- and red blood cell counts when compared to the wild-type mice (Rps19+/+, Pim-1+/+). Bone marrow cells in Rps19+/-, Pim-1-/- mice showed up-regulated levels of c-Myc and the anti-apoptotic factors Bcl2, Bcl-xl and Mcl-1 and reduced levels of the apoptotic factors Bak and Caspase 3 as well as the cell cycle regulator p21.

In summary, this thesis clarifies several mechanisms in the pathogenesis of DBA. Mutations in RPS19 results in coordinated down-regulation of several small subunit r-proteins causing haploinsufficiency for the small ribosomal subunit. RPS19 have multiple transcriptional start sites and mutations in the RPS19 5’UTR found in DBA patients result in reduced translational activity. At the cellular level, mutations in RPS19 and RPS24 cause distinct cell cycle defects and reduced cell proliferation. Finally, PIM-1 kinase and RPS19 cooperates in the proliferation of myeloid cells.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2009. 57 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 498
Keyword
Diamond-Blackfan anemia, RPS19, Ribosomal proteins, Haploinsufficiency, cell cycle, Apoptosis, Erythropoiesis
National Category
Medical Genetics
Research subject
Clinical Genetics
Identifiers
urn:nbn:se:uu:diva-110070 (URN)978-91-554-7655-7 (ISBN)
Public defence
2009-12-04, Fåhreussalen, C5, Dag Hammarskjölds Väg 20, Rudbeck Laboratory, Uppsala University, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2009-11-13 Created: 2009-11-02 Last updated: 2009-11-13Bibliographically approved
2. Molecular Studies of Diamond-Blackfan Anemia and Congenital Nail Dysplasia
Open this publication in new window or tab >>Molecular Studies of Diamond-Blackfan Anemia and Congenital Nail Dysplasia
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

The aim of this thesis is to investigate the effect of genetic mutations on the pathophysiology of two human disorders: Diamond-Blackfan Anemia (DBA) and isolated congenital nail dysplasia.

The first part of this thesis (Paper I-III) investigates the mechanism associated with DBA. DBA is a rare bone marrow failure syndrome characterized by the absence or decrease of erythroid precursor cells. The disease is further associated with growth retardation, malformations, predisposition to malignant disease and heterozygous mutations in ribosomal protein (RP) genes. The second part of this thesis (Paper IV) investigates the genetic basis of isolated autosomal recessive nail dysplasia characterized by pachyonychia and onycholysis of both finger- and toenails. It further dissects the molecular mechanisms regulating nail development.

In the first study, we investigated the previously reported RPS19/PIM-1 interaction by generating a combined Rps19/Pim-1 knockout mouse model. We found that allelic Rps19 insufficiency and Pim-1 deficiency have a cooperative effect on murine hematopoiesis resulting in increased myeloid cellularity associated with cell cycle alterations and reduced apoptosis. In the second study, we analyzed primary fibroblasts from DBA patients with truncating mutations in RPS19 or RPS24 and observed a marked delay in cellular growth associated with specific cell cycle defects. In the third study, we discovered that recombinant RPS19 binds its own mRNA and that the binding is altered when two DBA-associated RPS19 mutations are introduced. In the fourth study, we identified mutations in the WNT signaling receptor Frizzled 6 (FZD6). We observed that the nonsense mutant fails to interact with the first downstream effector Dishevelled. Fzd6 mutant mice displayed claw malformations and we detected a transient Fzd6 expression in the distal digits at the embryonic time point for nail development.

In summary, this thesis elucidates several mechanisms in the etiology of DBA and congenital nail dysplasia and mechanisms regulating nail development.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsalaiensis, 2010. 65 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 579
Keyword
Diamond-Blackfan Anemia, RPS19, RPS24, PIM-1, Erythropoiesis, Cell cycle, Apoptosis, Nail dysplasia, FZD6, WNT signaling
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-128067 (URN)978-91-554-7849-0 (ISBN)
Public defence
2010-09-17, Rudbecksalen, Rudbecklaboratoriet, Dag Hammarskjöldsv. 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-08-27 Created: 2010-07-19 Last updated: 2010-08-30

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