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Differential expression of RPS19 5’UTR variants implicated in Diamond-Blackfan anemia
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics. (Niklas Dahl)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics. (Niklas Dahl)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics. (Niklas Dahl)
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology, Medical Genetics. (Niklas Dahl)
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2012 (English)Article in journal (Other academic) In press
Abstract [en]

Heterozygous mutations in the ribosomal protein (RP) S19 gene RPS19 are found in about 25% of patients with the congenital erythroblastopenia Diamond-Blackfan anemia (DBA). The RPS19 gene encodes a single RPS19 isoform from three known transcriptional start sites (TSS) with different 5’ untranslated region (UTR). The regulation of RPS19 expression is poorly understood as well as the significance of different 5’UTRs. A few rare sequence variants within the 5’UTR have also been reported in patients with DBA. We determined the transcriptional start sites (TSS) and the tissue distribution of variant 5’UTRs of RPS19. Twenty-nine novel TSS in K562 cells and testis were identified. We then analyzed the relative proportion of three selected 5’UTRs of different length on a panel of primary tissues. The shorter 5’UTR were most abundant in all tissues but with large variations in relative levels of shorter versus longer transcripts. To clarify the effect of different RPS19 5’UTRs on translation we designed and expressed constructs using three 5’UTRs of different length. The short 5’UTR(+35nt.) translate 4-6 folds more efficiently than the two longer variants with 5’UTRs of 382nt. and 467nt., respectively We also introduced DBA associated insertion (c.-149_-148insGCCA, c.-149_-148insAGCC ) and deletion (c.-144_-141delTTTC) variants in the 5’UTR. . Interestingly, the DBA associated 5’UTR sequence variants showed a 20-30% reduction in RPS19 levels when compared to the corresponding w.t. constructs. Our results indicate that the RPS19 gene has a broad range of TSS with tissue specific variations. We also show that sequence variants in the 5’UTR in some DBA patients reduce RPS19 expression with implications for the pathophysiology of the disease.

Place, publisher, year, edition, pages
2012.
Keyword [en]
Diamond-Blackfan anemai, 5'UTR, translation, RPS19, haploinsufficiency
National Category
Medical Genetics
Research subject
Clinical Genetics
Identifiers
URN: urn:nbn:se:uu:diva-110063OAI: oai:DiVA.org:uu-110063DiVA: diva2:274868
Available from: 2009-11-02 Created: 2009-11-02 Last updated: 2012-04-24Bibliographically 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

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