Skarie JM, Link BA.. The primary open-angle glaucoma gene WDR36 functions in ribosomal RNA processing and interacts with the p53 stress-response pathway. Hum Mol Genet 17: 2474-2485

Department of Cell Biology, Neurobiology and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA.
Human Molecular Genetics (Impact Factor: 6.39). 06/2008; 17(16):2474-85. DOI: 10.1093/hmg/ddn147
Source: PubMed


Primary open-angle glaucoma (POAG) is a genetically complex neuropathy that affects retinal ganglion cells and is a leading
cause of blindness worldwide. WDR36, a gene of unknown function, was recently identified as causative for POAG at locus GLC1G. Subsequent studies found disease-associated
variants in control populations, leaving the role of WDR36 in this disease unclear. To address this issue, we determined the function of WDR36. We studied Wdr36 in zebrafish and found
it is the functional homolog of yeast Utp21. Utp21 is cell essential and functions in the nucleolar processing of 18S rRNA,
which is required for ribosome biogenesis. Evidence for functional homology comes from sequence alignment, ubiquitous expression,
sub-cellular localization to the nucleolus and loss-of-function phenotypes that include defects in 18S rRNA processing and
abnormal nucleolar morphology. Additionally, we show that loss of Wdr36 function leads to an activation of the p53 stress–response
pathway, suggesting that co-inheritance of defects in p53 pathway genes may influence the impact of WDR36 variants on POAG. Although these results overall do not provide evidence for or against a role of WDR36 in POAG, they do provide important baseline information for future studies.

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    • "Quantitative PCR results showed that in dg5nom1 larvae, p53 itself and its downstream genes expression were markedly increased. In some cases, phenotypes caused by ribosome biogenesis defect can be rescued by inhibition of p53 expression [23], [30], [31] but not in other [32]–[34]. In dg5nom1 mutant, neither the p53 MO nor the p53 null mutation could rescue the pancreatic defect, suggesting that p53 independent pathways are involved. "
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    ABSTRACT: Ribosome biogenesis is an important biological process for proper cellular function and development. Defects leading to improper ribosome biogenesis can cause diseases such as Diamond-Blackfan anemia and Shwachman-Bodian-Diamond syndrome. Nucleolar proteins are a large family of proteins and are involved in many cellular processes, including the regulation of ribosome biogenesis. Through a forward genetic screen and positional cloning, we identified and characterized a zebrafish line carrying mutation in nucleolar protein with MIF4G domain 1 (nom1), which encodes a conserved nulceolar protein with a role in pre-rRNA processing. Zebrafish nom1 mutants exhibit major defects in endoderm development, especially in exocrine pancreas. Further studies revealed that impaired proliferation of ptf1a-expressing pancreatic progenitor cells mainly contributed to the phenotype. RNA-seq and molecular analysis showed that ribosome biogenesis and pre-mRNA splicing were both affected in the mutant embryos. Several defects of ribosome assembly have been shown to have a p53-dependent mechanism. In the nom1 mutant, loss of p53 did not rescue the pancreatic defect, suggesting a p53-independent role. Further studies indicate that protein phosphatase 1 alpha, an interacting protein to Nom1, could partially rescue the pancreatic defect in nom1 morphants if a human nucleolar localization signal sequence was artificially added. This suggests that targeting Pp1α into the nucleolus by Nom1 is important for pancreatic proliferation. Altogether, our studies revealed a new mechanism involving Nom1 in controlling vertebrate exocrine pancreas formation.
    Full-text · Article · Jun 2014 · PLoS ONE
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    • "That the inhibition of rRNA transcription can lead to functional alterations of the nucleolus and upregulation of p53 protein has been demonstrated in many different ways including: genetic inactivation of the RNA polymerase I (Pol I) transcription factor TIF-1A [36], blockage of Pol I transcription factor UBF by microinjection of specific monoclonal antibodies [37], silencing the POLR1A gene coding for the Pol I catalytic subunit [38], treatment with the immunosuppressant mycophenolic acid [39] [40], low concentrations (b 10 nM) of actinomycin D, which intercalates into the GC-rich regions of rDNA [37] [41] or the small molecule compound CX-3543 (quarfloxin) that impairs binding of SL1/TIF-1B to the rDNA promoter leads to functional and morphological alterations of the nucleolus and stabilization of p53 protein levels [42]. Furthermore, the inhibition of rRNA processing by treatment with a chemotherapeutic compound 5-fluorouracil [43] [44] or decreased expression of proteins required for maturation of 18S and 28S rRNA such as hUTP18 [45], PAK1IP1[46], WDR3[47], WDR12 [45], WDR36 [48], nucleophosmin (NPM, B23) [49], nucleostemin [50] as well as specific RPs of either 40S or 60S [51] including RPS6 [51], RPS9 [52], RPL23 [53] [54] [55], RPL7a [51], RPS7 [51] [53] [54], RPL24 [31], RPL26 [53], RPL29 [56], RPL30 [56], RPL37 [57], RPS14 [58] [59], RPS19 [58], RPS15, RPS20 and RPL37 [60] can also induce a p53-mediated stress signal. Additionally , it has been recently demonstrated that the inhibition of RP nuclear import or nuclear export of ribosomal subunits by depletion of importin 7 (IPO7) or exportin 1 (XPO1), respectively, perturbs ribosome biogenesis , and consequently triggers the p53 response [61]. "
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    ABSTRACT: Errors in ribosome biogenesis can result in quantitative or qualitative defects in protein synthesis and consequently lead to improper execution of the genetic program and the development of specific diseases. Evidence has accumulated over the last decade suggesting that perturbation of ribosome biogenesis triggers a p53-activating checkpoint signaling pathway, often referred to as the ribosomal biogenesis stress checkpoint pathway. Although it was originally suggested that p53 has a prominent role in preventing diseases by monitoring the fidelity of ribosomal biogenesis, recent work has demonstrated that p53 activation upon impairment of ribosome biogenesis also mediates pathological manifestations in humans. Perturbations of ribosome biogenesis can trigger a p53-dependent checkpoint signaling pathway independently of DNA damage and the tumor suppressor ARF through inhibitory interactions of specific ribosomal components with the p53 negative regulator, Mdm2. Here we review the recent advances made towards understanding of this newly-recognized checkpoint signaling pathway, its role in health and disease, and discuss possible future directions in this exciting research field. This article is part of a Special Issue entitled: Role of the Nucleolus in Human Disease.
    Full-text · Article · Oct 2013 · Biochimica et Biophysica Acta
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    • "Accumulation of nuclear p53 leads to activation of cell cycle checkpoints, cell cycle arrest, and/or apoptosis. p53-mediated signaling is activated in ribosomopathy models [17,18], and p53 inhibition rescues craniofacial defects in a mouse model of Treacher Collins syndrome [19]. However, a recent zebrafish model of Shwachman-Diamond syndrome suggested that defects in pancreas and neutrophil development in this disorder occur independently of p53 [20]. "
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    ABSTRACT: North American Indian Childhood Cirrhosis (NAIC) is a rare, autosomal recessive, progressive cholestatic disease of infancy affecting the Cree-Ojibway first Nations of Quebec. All NAIC patients are homozygous for a missense mutation (R565W) in CIRH1A, the human homolog of the yeast nucleolar protein Utp4. Utp4 is part of the t-Utp subcomplex of the small subunit (SSU) processome, a ribonucleoprotein complex required for ribosomal RNA processing and small subunit assembly. NAIC has thus been proposed to be a primary ribosomal disorder (ribosomopathy); however, investigation of the pathophysiologic mechanism of this disease has been hindered by lack of an animal model. Here, using a morpholino oligonucleotide (MO)-based loss-of-function strategy, we have generated a model of NAIC in the zebrafish, Danio rerio. Zebrafish Cirhin shows substantial homology to the human homolog, and cirh1a mRNA is expressed in developing hepatocytes and biliary epithelial cells. Injection of two independent MOs directed against cirh1a at the one-cell stage causes defects in canalicular and biliary morphology in 5 dpf larvae. In addition, 5 dpf Cirhin-deficient larvae have dose-dependent defects in hepatobiliary function, as assayed by the metabolism of an ingested fluorescent lipid reporter. Previous yeast and in vitro studies have shown that defects in ribosome biogenesis cause stabilization and nuclear accumulation of p53, which in turn causes p53-mediated cell cycle arrest and/or apoptosis. Thus, the nucleolus appears to function as a cellular stress sensor in some cell types. In accordance with this hypothesis, transcriptional targets of p53 are upregulated in Cirhin-deficient zebrafish embryos, and defects in biliary function seen in Cirhin-deficient larvae are completely abrogated by mutation of tp53. Our data provide the first in vivo evidence of a role for Cirhin in biliary development, and support the hypothesis that congenital defects affecting ribosome biogenesis can activate a cellular stress response mediated by p53.
    Full-text · Article · Oct 2013 · PLoS ONE
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