Posttranscriptional down‐regulation of small ribosomal subunit proteins correlates with reduction of 18S rRNA in RPS19 deficiency

Badhai, Jitendra; Fröjmark, Anne-Sophie; Razzaghian, Hamid Reza; Davey, Edward J.; Schuster, Jens; Dahl, Niklas

doi:10.1016/j.febslet.2009.05.023

Cited by 20 publications

(26 citation statements)

References 25 publications

(38 reference statements)

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“…The observed concomitant reduction of both small subunit proteins in mutant cell lines may be explained by a deficient assembly of the small subunit when one component is present in insufficient amounts. A similar coordinated down regulation of ribosomal proteins from the same subunit was recently reported in HeLa cells subject to siRNA knock-down of specific RPs [26] and in patient derived cell-lines [27]. …”

Section: Resultssupporting

confidence: 72%

Ribosomal protein S19 and S24 insufficiency cause distinct cell cycle defects in Diamond–Blackfan anemia

Badhai

Fröjmark

Davey

et al. 2009

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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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 fibroblast 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.

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Section: Resultssupporting

confidence: 72%

Ribosomal protein S19 and S24 insufficiency cause distinct cell cycle defects in Diamond–Blackfan anemia

Badhai

Fröjmark

Davey

et al. 2009

Biochimica et Biophysica Acta (BBA) - Molecular Basis of Diseas

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“…The cell suspension was centrifuged for 10 min at +4 degrees Celsius at 13000 rpm to remove cell debris and the lysate was stored at −70 degrees Celsius until use [34]. Lysates were separated on Bis-Tris SDS-PAGE gels (Invitrogen), transferred to PVDF membranes (Millipore) and incubated with primary and secondary antibodies as described previously [34].…”

Section: Methodsmentioning

confidence: 99%

siRNA Silencing of Proteasome Maturation Protein (POMP) Activates the Unfolded Protein Response and Constitutes a Model for KLICK Genodermatosis

et al. 2012

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Keratosis linearis with ichthyosis congenita and keratoderma (KLICK) is an autosomal recessive skin disorder associated with a single-nucleotide deletion in the 5′untranslated region of the proteasome maturation protein (POMP) gene. The deletion causes a relative switch in transcription start sites for POMP, predicted to decrease levels of POMP protein in terminally differentiated keratinocytes. To investigate the pathophysiology behind KLICK we created an in vitro model of the disease using siRNA silencing of POMP in epidermal air-liquid cultures. Immunohistochemical analysis of the tissue constructs revealed aberrant staining of POMP, proteasome subunits and the skin differentiation marker filaggrin when compared to control tissue constructs. The staining patterns of POMP siRNA tissue constructs showed strong resemblance to those observed in skin biopsies from KLICK patients. Western blot analysis of lysates from the organotypic tissue constructs revealed an aberrant processing of profilaggrin to filaggrin in samples transfected with siRNA against POMP. Knock-down of POMP expression in regular cell cultures resulted in decreased amounts of proteasome subunits. Prolonged silencing of POMP in cultured cells induced C/EBP homologous protein (CHOP) expression consistent with an activation of the unfolded protein response and increased endoplasmic reticulum (ER) stress. The combined results indicate that KLICK is caused by reduced levels of POMP, leading to proteasome insufficiency in differentiating keratinocytes. Proteasome insufficiency disturbs terminal epidermal differentiation, presumably by increased ER stress, and leads to perturbed processing of profilaggrin. Our findings underline a critical role for the proteasome in human epidermal differentiation.

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“…Lysates were separated on Bis-Tris SDS-PAGE gels (Invitrogen), transferred to PVDF membranes (Millipore AB, Solna, Sweden) and incubated with primary and secondary antibodies as described previously [3]. Secondary antibodies were conjugated with Alexa Fluor 680 (anti-mouse; Molecular probes/Invitrogen) or IRDye 800 (anti-rabbit; Li-Cor Bioscience, Cambridge, UK) for visualization with Odyssey infrared imaging system Ò (LiCor Bioscience).…”

Section: Immunoblot Analysismentioning

confidence: 99%

“…For western blot analysis primary keratinocytes from three healthy individuals and patient 1were harvested by trypsinization and protein lysates were prepared as described elsewhere [3]. Lysates were separated on Bis-Tris SDS-PAGE gels (Invitrogen), transferred to PVDF membranes (Millipore AB, Solna, Sweden) and incubated with primary and secondary antibodies as described previously [3].…”

Section: Immunoblot Analysismentioning

confidence: 99%

Ichthyin/NIPAL4 localizes to keratins and desmosomes in epidermis and Ichthyin mutations affect epidermal lipid metabolism

et al. 2012

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Autosomal recessive congenital ichthyosis (ARCI) is a group of disorders characterized by abnormal desquamation of the skin and a disrupted epidermal water barrier. Ichthyin/NIPAL4 gene mutations have been identified in a subgroup of ARCI patients, but the role of ichthyin in epidermis remains elusive. In order to obtain new insights concerning the characteristics of ichthyin and the ARCI pathogenesis, we studied the expression and localization of ichthyin and related epidermal components in cultured keratinocytes and skin sections from patients with Ichthyin mutations and healthy controls. We observed an up-regulation of Ichthyin mRNA levels after in vitro differentiation of keratinocytes from both a patient with Ichthyin mutations and controls. Confocal and electron microscopy analyses of immunolabeled skin sections revealed that ichthyin localizes to desmosomes and keratins in both patients with mutant Ichthyin and controls, with an increased immunolabeling in patients. Nile red lipid analysis of skin sections exposed intra-cellular lipid accumulations in cells of the granular and cornified layers in patients but not in controls, consistent with the pathognomonic lipid membrane structures previously identified in epidermis from patients. Our combined findings indicate that ichthyin is associated with keratins and desmosomes in epidermis and is involved in lipid metabolism, possibly through processing of lamellar bodies. These results provide new clues to the understanding of the epidermal water barrier and the pathogenesis in ARCI.

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Posttranscriptional down‐regulation of small ribosomal subunit proteins correlates with reduction of 18S rRNA in RPS19 deficiency

Cited by 20 publications

References 25 publications

Ribosomal protein S19 and S24 insufficiency cause distinct cell cycle defects in Diamond–Blackfan anemia

Ribosomal protein S19 and S24 insufficiency cause distinct cell cycle defects in Diamond–Blackfan anemia

siRNA Silencing of Proteasome Maturation Protein (POMP) Activates the Unfolded Protein Response and Constitutes a Model for KLICK Genodermatosis

Ichthyin/NIPAL4 localizes to keratins and desmosomes in epidermis and Ichthyin mutations affect epidermal lipid metabolism

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