Correction: WRAP53 Is Essential for Cajal Body Formation and for Targeting the Survival of Motor Neuron Complex to Cajal Bodies.
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ABSTRACT:
SUBMITTER: Mahmoudi S
PROVIDER: S-EPMC6155438 | biostudies-literature | 2018 Sep
REPOSITORIES: biostudies-literature
Publications
Correction: WRAP53 Is Essential for Cajal Body Formation and for Targeting the Survival of Motor Neuron Complex to Cajal Bodies.
Mahmoudi Salah S Henriksson Sofia S Weibrecht Irene I Smith Stephen S Söderberg Ola O Strömblad Staffan S Wiman Klas G KG Farnebo Marianne M
PLoS biology 20180925 9
[This corrects the article DOI: 10.1371/journal.pbio.1000521.]. ...[more]
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Project description:The WRAP53 gene gives rise to a p53 antisense transcript that regulates p53. This gene also encodes a protein that directs small Cajal body-specific RNAs to Cajal bodies. Cajal bodies are nuclear organelles involved in diverse functions such as processing ribonucleoproteins important for splicing. Here we identify the WRAP53 protein as an essential factor for Cajal body maintenance and for directing the survival of motor neuron (SMN) complex to Cajal bodies. By RNA interference and immunofluorescence we show that Cajal bodies collapse without WRAP53 and that new Cajal bodies cannot be formed. By immunoprecipitation we find that WRAP53 associates with the Cajal body marker coilin, the splicing regulatory protein SMN, and the nuclear import receptor importin?, and that WRAP53 is essential for complex formation between SMN-coilin and SMN-importin?. Furthermore, depletion of WRAP53 leads to accumulation of SMN in the cytoplasm and prevents the SMN complex from reaching Cajal bodies. Thus, WRAP53 mediates the interaction between SMN and associated proteins, which is important for nuclear targeting of SMN and the subsequent localization of the SMN complex to Cajal bodies. Moreover, we detect reduced WRAP53-SMN binding in patients with spinal muscular atrophy, which is the leading genetic cause of infant mortality worldwide, caused by mutations in SMN1. This suggests that loss of WRAP53-mediated SMN trafficking contributes to spinal muscular atrophy.
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Project description:Low levels of the survival motor neuron (SMN) protein cause spinal muscular atrophy, the leading genetic disorder for infant mortality. SMN is ubiquitously expressed in various cell types and localizes in both the cytoplasm and the nucleus, where it concentrates in two subnuclear structures termed Cajal body (CB) and gems. In addition, SMN can also be detected in the nucleolus of neurons. Mechanisms that control SMN sorting in the cell remain largely unknown. Here, we report that the ubiquitin (Ub) ligase Itch directly interacts with and monoubiquitinates SMN. Monoubiquitination of SMN has a mild effect on promoting proteasomal degradation of SMN. We generated two SMN mutants, SMN(K0), in which all lysines are mutated to arginines and thereby abolishing SMN ubiquitination, and Ub-SMN(K0), in which a single Ub moiety is fused at the N-terminus of SMN(K0) and thereby mimicking SMN monoubiquitination. Immunostaining assays showed that SMN(K0) mainly localizes in the nucleus, whereas Ub-SMN(K0) localizes in both the cytoplasm and the nucleolus in neuronal SH-SY5Y cells. Interestingly, canonical CB foci and coilin/small nuclear ribonucleoprotein (snRNP) co-localization are significantly impaired in SH-SY5Y cells stably expressing SMN(K0) or Ub-SMN(K0). Thus, our studies discover that Itch monoubiquitinates SMN and monoubiquitination of SMN plays an important role in regulating its cellular localization. Moreover, mislocalization of SMN disrupts CB integrity and likely impairs snRNP maturation.
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Project description:Mutation of the survival motor neurons 1 (SMN1) gene causes motor neuron apoptosis and represents the major cause of spinal muscular atrophy in humans. Biochemical studies have established that the SMN protein plays an important role in spliceosomal small nuclear ribonucleoprotein (snRNP) biogenesis and that the SMN complex can interact with the zinc finger protein ZPR1. Here we report that targeted ablation of the Zpr1 gene in mice disrupts the subcellular localization of both SMN and spliceosomal snRNPs. Specifically, SMN localization to Cajal bodies and gems was not observed in cells derived from Zpr1-/- embryos and the amount of cytoplasmic snRNP detected in Zpr1-/- embryos was reduced compared with that in wild-type embryos. We found that Zpr1-/- mice die during early embryonic development, with reduced proliferation and increased apoptosis. These effects of Zpr1 gene disruption were confirmed and extended in studies of cultured motor neuron-like cells using small interfering RNA-mediated Zpr1 gene suppression; ZPR1 deficiency caused growth cone retraction, axonal defects, and apoptosis. Together, these data indicate that ZPR1 contributes to the regulation of SMN complexes and that it is essential for cell survival.
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Project description:Cajal bodies are specialized and dynamic compartments in the nucleus that are involved in the biogenesis of small nuclear ribonucleoproteins (snRNPs). Because of the dynamic and varied roles of Cajal bodies, it is of great interest to identify the components of Cajal bodies to better understand their functions. We performed a genome-wide screen to identify proteins that colocalize with coilin, the marker protein of Cajal bodies. In this study, we identified and characterized Fam118B as a newly discovered component of Cajal bodies. Fam118B is widely expressed in a variety of cell lines derived from various origins. Overexpression of Fam118B changes the canonical morphology of Cajal bodies, whereas depletion of Fam118B disrupts the localization of components of Cajal bodies, including coilin, the survival of motor neuron protein (SMN) and the Sm protein D1 (SmD1, also known as SNRPD1). Moreover, depletion of Fam118B reduces splicing capacity and inhibits cell proliferation. In addition, Fam118B associates with coilin and SMN proteins. Fam118B depletion reduces symmetric dimethylarginine modification of SmD1, which in turn diminishes the binding of SMN to this Sm protein. Taken together, these data indicate that Fam118B, by regulating SmD1 symmetric dimethylarginine modification, plays an important role in Cajal body formation, snRNP biogenesis and cell viability.
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Project description:Cajal bodies are small nuclear organelles with a number of nuclear functions. Here we show that FLICE-associated huge protein (FLASH), originally described as a component of the apoptosis signaling pathway, is mainly localized in Cajal bodies and is essential for their structure. Reduction in FLASH expression by short hairpin RNA results in disruption of the normal architecture of the Cajal body and relocalization of its components. Because the function of FLASH in the apoptosis receptor signaling pathway has been strongly questioned, we have now identified a clear function for this protein.
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Project description:Approximately half of all cases of Hoyeraal-Hreidarsson syndrome (HHS), a multisystem disorder characterized by bone marrow failure, developmental defects and very short telomeres, are caused by germline mutations in genes related to telomere biology. However, the varying symptoms and severity of the disease indicate that additional mechanisms are involved. Here, a 3-year-old boy with HHS was found to carry biallelic germline mutations in WRAP53 (WD40 encoding RNA antisense to p53), that altered two highly conserved amino acids (L283F and R398W) in the WD40 scaffold domain of the protein encoded. WRAP53β (also known as TCAB1 or WDR79) is involved in intracellular trafficking of telomerase, Cajal body functions and DNA repair. We found that both mutations cause destabilization, mislocalization and faulty interactions of WRAP53β, defects linked to misfolding by the TRiC chaperonin complex. Consequently, WRAP53β HHS mutants cannot elongate telomeres, maintain Cajal bodies or repair DNA double-strand breaks. These findings provide a molecular explanation for the pathogenesis underlying WRAP53β-associated HHS and highlight the potential contribution of DNA damage and/or defects in Cajal bodies to the early onset and/or severity of this disease.
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Project description:During their biogenesis small nuclear RNAs (snRNAs) undergo multiple covalent modifications that require guide RNAs to direct methylase and pseudouridylase enzymes to the appropriate nucleotides. Because of their localization in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific RNAs (scaRNAs). Using a fluorescent primer extension technique, we mapped the modified nucleotides in Drosophila U1, U2, U4, and U5 snRNAs. By fluorescent in situ hybridization (FISH) we showed that seven Drosophila scaRNAs are concentrated in easily detectable CBs. We used two assays based on Xenopus oocyte nuclei to demonstrate that three of these Drosophila scaRNAs do, in fact, function as guide RNAs. In flies null for the CB marker protein coilin, CBs are absent and there are no localized FISH signals for the scaRNAs. Nevertheless, biochemical experiments show that scaRNAs are present at normal levels and snRNAs are properly modified. Our experiments demonstrate that several scaRNAs are concentrated as expected in the CBs of wild-type Drosophila, but they function equally well in the nucleoplasm of mutant flies that lack CBs. We propose that the snRNA modification machinery is not limited to CBs, but is dispersed throughout the nucleoplasm of cells in general.
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Project description:Small nuclear ribonucleoproteins (snRNPs) play a fundamental role in pre-mRNA processing in the nucleus. The biogenesis of snRNPs involves a sequence of events that occurs in both the nucleus and cytoplasm. Despite the wealth of biochemical information about the cytoplasmic assembly of snRNPs, little is known about the spatial organization of snRNPs in the cytoplasm. In the cytoplasm of larch microsporocytes, a cyclic appearance of bodies containing small nuclear RNA (snRNA) and Sm proteins was observed during anther meiosis. We observed a correlation between the occurrence of cytoplasmic snRNP bodies, the levels of Sm proteins, and the dynamic formation of Cajal bodies. Larch microsporocytes were used for these studies. This model is characterized by natural fluctuations in the level of RNA metabolism, in which periods of high transcriptional activity are separated from periods of low transcriptional activity. In designing experiments, the authors considered the differences between the nuclear and cytoplasmic phases of snRNP maturation and generated a hypothesis about the direct participation of Sm proteins in a molecular switch triggering the formation of Cajal bodies.
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Project description:Amyotrophic lateral sclerosis (ALS) is a heterogeneous degenerative motor neuron disease linked to numerous genetic mutations in apparently unrelated proteins. These proteins, including SOD1, TDP-43, and FUS, are highly aggregation-prone and form a variety of intracellular inclusion bodies that are characteristic of different neuropathological subtypes of the disease. Contained within these inclusions are a variety of proteins that do not share obvious characteristics other than coaggregation. However, recent evidence from other neurodegenerative disorders suggests that disease-affected biochemical pathways can be characterized by the presence of proteins that are supersaturated, with cellular concentrations significantly greater than their solubilities. Here, we show that the proteins that form inclusions of mutant SOD1, TDP-43, and FUS are not merely a subset of the native interaction partners of these three proteins, which are themselves supersaturated. To explain the presence of coaggregating proteins in inclusions in the brain and spinal cord, we observe that they have an average supersaturation even greater than the average supersaturation of the native interaction partners in motor neurons, but not when scores are generated from an average of other human tissues. These results suggest that inclusion bodies in various forms of ALS result from a set of proteins that are metastable in motor neurons, and thus prone to aggregation upon a disease-related progressive collapse of protein homeostasis in this specific setting.
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