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1.
EMBO Mol Med ; 11(7): e10201, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31273937

RESUMO

PARN, poly(A)-specific ribonuclease, regulates the turnover of mRNAs and the maturation and stabilization of the hTR RNA component of telomerase. Biallelic PARN mutations were associated with Høyeraal-Hreidarsson (HH) syndrome, a rare telomere biology disorder that, because of its severity, is likely not exclusively due to hTR down-regulation. Whether PARN deficiency was affecting the expression of telomere-related genes was still unclear. Using cells from two unrelated HH individuals carrying novel PARN mutations and a human PARN knock-out (KO) cell line with inducible PARN complementation, we found that PARN deficiency affects both telomere length and stability and down-regulates the expression of TRF1, TRF2, TPP1, RAP1, and POT1 shelterin transcripts. Down-regulation of dyskerin-encoding DKC1 mRNA was also observed and found to result from p53 activation in PARN-deficient cells. We further showed that PARN deficiency compromises ribosomal RNA biogenesis in patients' fibroblasts and cells from heterozygous Parn KO mice. Homozygous Parn KO however resulted in early embryonic lethality that was not overcome by p53 KO. Our results refine our knowledge on the pleiotropic cellular consequences of PARN deficiency.

2.
EMBO Mol Med ; 2019 Jun 06.
Artigo em Inglês | MEDLINE | ID: mdl-31171653

RESUMO

PARN, poly(A)-specific ribonuclease, regulates the turnover of mRNAs and the maturation and stabilization of the hTR RNA component of telomerase. Biallelic PARN mutations were associated with Høyeraal-Hreidarsson (HH) syndrome, a rare telomere biology disorder that, because of its severity, is likely not exclusively due to hTR down-regulation. Whether PARN deficiency was affecting the expression of telomere-related genes was still unclear. Using cells from two unrelated HH individuals carrying novel PARN mutations and a human PARN knock-out (KO) cell line with inducible PARN complementation, we found that PARN deficiency affects both telomere length and stability and down-regulates the expression of TRF1, TRF2, TPP1, RAP1, and POT1 shelterin transcripts. Down-regulation of dyskerin-encoding DKC1 mRNA was also observed and found to result from p53 activation in PARN-deficient cells. We further showed that PARN deficiency compromises ribosomal RNA biogenesis in patients' fibroblasts and cells from heterozygous Parn KO mice. Homozygous Parn KO however resulted in early embryonic lethality that was not overcome by p53 KO. Our results refine our knowledge on the pleiotropic cellular consequences of PARN deficiency.

3.
PLoS Genet ; 15(2): e1007917, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30707697

RESUMO

Hbs1 has been established as a central component of the cell's translational quality control pathways in both yeast and prokaryotic models; however, the functional characteristics of its human ortholog (Hbs1L) have not been well-defined. We recently reported a novel human phenotype resulting from a mutation in the critical coding region of the HBS1L gene characterized by facial dysmorphism, severe growth restriction, axial hypotonia, global developmental delay and retinal pigmentary deposits. Here we further characterize downstream effects of the human HBS1L mutation. HBS1L has three transcripts in humans, and RT-PCR demonstrated reduced mRNA levels corresponding with transcripts V1 and V2 whereas V3 expression was unchanged. Western blot analyses revealed Hbs1L protein was absent in the patient cells. Additionally, polysome profiling revealed an abnormal aggregation of 80S monosomes in patient cells under baseline conditions. RNA and ribosomal sequencing demonstrated an increased translation efficiency of ribosomal RNA in Hbs1L-deficient fibroblasts, suggesting that there may be a compensatory increase in ribosome translation to accommodate the increased 80S monosome levels. This enhanced translation was accompanied by upregulation of mTOR and 4-EBP protein expression, suggesting an mTOR-dependent phenomenon. Furthermore, lack of Hbs1L caused depletion of Pelota protein in both patient cells and mouse tissues, while PELO mRNA levels were unaffected. Inhibition of proteasomal function partially restored Pelota expression in human Hbs1L-deficient cells. We also describe a mouse model harboring a knockdown mutation in the murine Hbs1l gene that shared several of the phenotypic elements observed in the Hbs1L-deficient human including facial dysmorphism, growth restriction and retinal deposits. The Hbs1lKO mice similarly demonstrate diminished Pelota levels that were rescued by proteasome inhibition.


Assuntos
Proteínas de Ligação ao GTP/genética , Mamíferos/genética , Proteínas dos Microfilamentos/genética , Monossomia/genética , Animais , Linhagem Celular , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Mutação/genética , Fenótipo , Polirribossomos/genética , Complexo de Endopeptidases do Proteassoma/genética , RNA/genética , RNA Mensageiro/genética , Ribossomos/genética , Serina-Treonina Quinases TOR/genética , Regulação para Cima/genética
5.
Am J Hum Genet ; 2018 Nov 21.
Artigo em Inglês | MEDLINE | ID: mdl-30503522

RESUMO

Diamond-Blackfan anemia (DBA) is a rare bone marrow failure disorder that affects 7 out of 1,000,000 live births and has been associated with mutations in components of the ribosome. In order to characterize the genetic landscape of this heterogeneous disorder, we recruited a cohort of 472 individuals with a clinical diagnosis of DBA and performed whole-exome sequencing (WES). We identified relevant rare and predicted damaging mutations for 78% of individuals. The majority of mutations were singletons, absent from population databases, predicted to cause loss of function, and located in 1 of 19 previously reported ribosomal protein (RP)-encoding genes. Using exon coverage estimates, we identified and validated 31 deletions in RP genes. We also observed an enrichment for extended splice site mutations and validated their diverse effects using RNA sequencing in cell lines obtained from individuals with DBA. Leveraging the size of our cohort, we observed robust genotype-phenotype associations with congenital abnormalities and treatment outcomes. We further identified rare mutations in seven previously unreported RP genes that may cause DBA, as well as several distinct disorders that appear to phenocopy DBA, including nine individuals with biallelic CECR1 mutations that result in deficiency of ADA2. However, no new genes were identified at exome-wide significance, suggesting that there are no unidentified genes containing mutations readily identified by WES that explain >5% of DBA-affected case subjects. Overall, this report should inform not only clinical practice for DBA-affected individuals, but also the design and analysis of rare variant studies for heterogeneous Mendelian disorders.

6.
Wiley Interdiscip Rev RNA ; : e1516, 2018 Nov 08.
Artigo em Inglês | MEDLINE | ID: mdl-30406965

RESUMO

The synthesis of ribosomal subunits in eukaryotes requires the interplay of numerous maturation and assembly factors (AFs) that intervene in the insertion of ribosomal proteins within pre-ribosomal particles, the ribosomal subunit precursors, as well as in pre-ribosomal RNA (rRNA) processing and folding. Here, we review the intricate nuclear and cytoplasmic maturation steps of pre-40S particles, the precursors to the small ribosomal subunits, in both yeast and human cells, with particular emphasis on the timing and mechanisms of AF association with and dissociation from pre-40S particles and the roles of these AFs in the maturation process. We highlight the particularly complex pre-rRNA processing pathway in human cells, compared to yeast, to generate the mature 18S rRNA. We discuss the information gained from the recently published cryo-electron microscopy atomic models of yeast and human pre-40S particles, as well as the checkpoint/quality control systems that seem to operate to probe functional sites within yeast cytoplasmic pre-40S particles. This article is categorized under: RNA Processing > rRNA Processing Translation > Ribosome Biogenesis.

7.
Biomolecules ; 8(4)2018 Oct 24.
Artigo em Inglês | MEDLINE | ID: mdl-30356013

RESUMO

Ribosomal RNAs, the most abundant cellular RNA species, have evolved as the structural scaffold and the catalytic center of protein synthesis in every living organism. In eukaryotes, they are produced from a long primary transcript through an intricate sequence of processing steps that include RNA cleavage and folding and nucleotide modification. The mechanisms underlying this process in human cells have long been investigated, but technological advances have accelerated their study in the past decade. In addition, the association of congenital diseases to defects in ribosome synthesis has highlighted the central place of ribosomal RNA maturation in cell physiology regulation and broadened the interest in these mechanisms. Here, we give an overview of the current knowledge of pre-ribosomal RNA processing in human cells in light of recent progress and discuss how dysfunction of this pathway may contribute to the physiopathology of congenital diseases.

8.
PLoS Genet ; 14(3): e1007226, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29518074

RESUMO

Gene expression in a tissue-specific context depends on the combined efforts of epigenetic, transcriptional and post-transcriptional processes that lead to the production of specific proteins that are important determinants of cellular identity. Ribosomes are a central component of the protein biosynthesis machinery in cells; however, their regulatory roles in the translational control of gene expression in skeletal muscle remain to be defined. In a genetic screen to identify critical regulators of myogenesis, we identified a DEAD-Box RNA helicase, DDX27, that is required for skeletal muscle growth and regeneration. We demonstrate that DDX27 regulates ribosomal RNA (rRNA) maturation, and thereby the ribosome biogenesis and the translation of specific transcripts during myogenesis. These findings provide insight into the translational regulation of gene expression in myogenesis and suggest novel functions for ribosomes in regulating gene expression in skeletal muscles.


Assuntos
RNA Helicases DEAD-box/metabolismo , Músculo Esquelético/fisiologia , Biossíntese de Proteínas , RNA Ribossômico/metabolismo , Animais , Animais Geneticamente Modificados , Linhagem Celular , Nucléolo Celular/metabolismo , Nucléolo Celular/ultraestrutura , Proliferação de Células/genética , RNA Helicases DEAD-box/genética , Embrião não Mamífero , Camundongos , Desenvolvimento Muscular/fisiologia , Músculo Esquelético/citologia , Músculo Esquelético/crescimento & desenvolvimento , Mioblastos/citologia , Mioblastos/fisiologia , Fator de Transcrição PAX2/genética , Fator de Transcrição PAX2/metabolismo , RNA Ribossômico/genética , Regeneração/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
9.
Haematologica ; 103(6): 949-958, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29599205

RESUMO

Diamond-Blackfan anemia (DBA) is a rare inherited bone marrow failure disorder linked predominantly to ribosomal protein gene mutations. Here the European DBA consortium reports novel mutations identified in the RPL15 gene in 6 unrelated individuals diagnosed with DBA. Although point mutations have not been previously reported for RPL15, we identified 4 individuals with truncating mutations p.Tyr81* (in 3 of 4) and p.Gln29*, and 2 with missense variants p.Leu10Pro and p.Lys153Thr. Notably, 75% (3 of 4) of truncating mutation carriers manifested with severe hydrops fetalis and required intrauterine transfusions. Even more remarkable is the observation that the 3 carriers of p.Tyr81* mutation became treatment-independent between four and 16 months of life and maintained normal blood counts until their last follow up. Genetic reversion at the DNA level as a potential mechanism of remission was not observed in our patients. In vitro studies revealed that cells carrying RPL15 mutations have pre-rRNA processing defects, reduced 60S ribosomal subunit formation, and severe proliferation defects. Red cell culture assays of RPL15-mutated primary erythroblast cells also showed a severe reduction in cell proliferation, delayed erythroid differentiation, elevated TP53 activity, and increased apoptosis. This study identifies a novel subgroup of DBA with mutations in the RPL15 gene with an unexpected high rate of hydrops fetalis and spontaneous, long-lasting remission.

10.
Eur J Med Genet ; 61(11): 664-673, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-29081386

RESUMO

Diamond-Blackfan anemia (DBA) is a rare congenital erythroblastopenia and inherited bone marrow failure syndrome that affects approximately seven individuals in every million live births. In addition to anemia, about 50% of all DBA patients suffer from various physical malformations of the face, hands, heart, or urogenital region. The disorder is almost exclusively driven by haploinsufficient mutations in one of several ribosomal protein (RP) genes, although for ∼30% of diagnosed patients no mutation is found in any of the known DBA-linked genes. Because DBA is such a rare disease with a particularly wide range of clinical phenotypes and molecular signatures, the development of collaborative efforts such as the ERARE-funded European DBA consortium (EuroDBA) has become imperative for DBA research. EuroDBA was founded in 2012 and brings together dedicated clinical and biological researchers of DBA from France, Italy, the Netherlands, Germany, Israel, Poland, and Turkey to achieve a number of goals including the consolidation of data in patient registries, establishment of minimal diagnostic criteria, and projects aimed at more fully describing the different mutations linked to DBA. This review will cover the history of the EuroDBA registries, the methods used by EuroDBA in the diagnosis of DBA, and how the consortium has successfully worked together towards the discovery of new DBA-linked genes and the better understanding their pathophysiological effects.

11.
Biochem Biophys Res Commun ; 495(2): 1839-1845, 2018 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-29225165

RESUMO

Mutations in genes encoding ribosomal proteins have been identified in Diamond-Blackfan anemia (DBA), a rare genetic disorder that presents with a prominent erythroid phenotype. TP53 has been implicated in the pathophysiology of DBA with ribosomal protein (RP) L11 playing a crucial role in the TP53 response. Interestingly, RPL11 also controls the transcriptional activity of c-Myc, an oncoprotein that positively regulates ribosome biogenesis. In the present study, we analyzed the consequences of rpl11 depletion on erythropoiesis and ribosome biogenesis in zebrafish. As expected, Rpl11-deficient zebrafish exhibited defects in ribosome biogenesis and an anemia phenotype. However, co-inhibition of Tp53 did not alleviate the erythroid aplasia in these fish. Next, we explored the role of c-Myc in RPL11-deficient cellular and animal models. c-Myc and its target nucleolar proteins showed upregulation and increased localization in the head region of Rpl11-deficient zebrafish, where the morphological abnormalities and tp53 expression were more pronounced. Interestingly, in blood cells derived from DBA patients with mutations in RPL11, the biogenesis of ribosomes was defective, but the expression level of c-Myc and its target nucleolar proteins was unchanged. The results suggest a model whereby RPL11 deficiency activates the synthesis of c-Myc target nucleolar proteins, which subsequently triggers a p53 response. These results further demonstrate that the induction of Tp53 mediates the morphological, but not erythroid, defects associated with RPL11 deficiency.


Assuntos
Anemia de Diamond-Blackfan/fisiopatologia , Proteínas Ribossômicas/deficiência , Anemia de Diamond-Blackfan/genética , Anemia de Diamond-Blackfan/patologia , Animais , Modelos Animais de Doenças , Eritropoese/genética , Proteínas de Peixes/deficiência , Proteínas de Peixes/genética , Genes myc , Genes p53 , Humanos , Mutação , Processamento Pós-Transcricional do RNA , Proteínas Ribossômicas/genética , Peixe-Zebra
12.
PLoS One ; 12(11): e0187977, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29190286

RESUMO

The nucleolus produces the large polycistronic transcript (47S precursor) containing the 18S, 5.8S and 28S rRNA sequences and hosts most of the nuclear steps of pre-rRNA processing. Among numerous components it contains condensed chromatin and active rRNA genes which adopt a more accessible conformation. For this reason, it is a paradigm of chromosome territory organization. Active rRNA genes are clustered within several fibrillar centers (FCs), in which they are maintained in an open configuration by Upstream Binding Factor (UBF) molecules. Here, we used the reproducible reorganization of nucleolar components induced by the inhibition of rRNA synthesis by Actinomycin D (AMD) to address the steps of the spatiotemporal reorganization of FCs and nucleolar condensed chromatin. To reach that goal, we used two complementary approaches: i) time-lapse confocal imaging of cells expressing one or several GFP-tagged proteins (fibrillarin, UBF, histone H2B) and ii) ultrastructural identification of nucleolar components involved in the reorganization. Data obtained by time lapse confocal microscopy were analyzed through detailed 3D imaging. This allowed us to demonstrate that AMD treatment induces no fusion and no change in the relative position of the different nucleoli contained in one nucleus. In contrast, for each nucleolus, we observed step by step gathering and fusion of both FCs and nucleolar condensed chromatin. To analyze the reorganization of FCs and condensed chromatin at a higher resolution, we performed correlative light and electron microscopy electron microscopy (CLEM) imaging of the same cells. We demonstrated that threads of intranucleolar condensed chromatin are localized in a complex 3D network of vacuoles. Upon AMD treatment, these structures coalesce before migrating toward the perinucleolar condensed chromatin, to which they finally fuse. During their migration, FCs, which are all linked to ICC, are pulled by the latter to gather as caps disposed at the periphery of nucleoli.


Assuntos
Compartimento Celular , Nucléolo Celular/metabolismo , Cromatina/metabolismo , RNA Ribossômico/antagonistas & inibidores , Linhagem Celular Tumoral , Dactinomicina/farmacologia , Humanos , Microscopia Eletrônica de Transmissão , RNA Ribossômico/biossíntese , RNA Ribossômico/efeitos dos fármacos
13.
Nucleic Acids Res ; 45(11): 6822-6836, 2017 Jun 20.
Artigo em Inglês | MEDLINE | ID: mdl-28402503

RESUMO

The poly-A specific ribonuclease (PARN), initially characterized for its role in mRNA catabolism, supports the processing of different types of non-coding RNAs including telomerase RNA. Mutations in PARN are linked to dyskeratosis congenita and pulmonary fibrosis. Here, we show that PARN is part of the enzymatic machinery that matures the human 18S ribosomal RNA (rRNA). Consistent with its nucleolar steady-state localization, PARN is required for 40S ribosomal subunit production and co-purifies with 40S subunit precursors. Depletion of PARN or expression of a catalytically-compromised PARN mutant results in accumulation of 3΄ extended 18S rRNA precursors. Analysis of these processing intermediates reveals a defect in 3΄ to 5΄ trimming of the internal transcribed spacer 1 (ITS1) region, subsequent to endonucleolytic cleavage at site E. Consistent with a function of PARN in exonucleolytic trimming of 18S-E pre-rRNA, recombinant PARN can process the corresponding ITS1 RNA fragment in vitro. Trimming of 18S-E pre-rRNA by PARN occurs in the nucleus, upstream of the final endonucleolytic cleavage by the endonuclease NOB1 in the cytoplasm. These results identify PARN as a new component of the ribosome biogenesis machinery in human cells. Defects in ribosome biogenesis could therefore underlie the pathologies linked to mutations in PARN.


Assuntos
Exorribonucleases/fisiologia , RNA Ribossômico 18S/biossíntese , Núcleo Celular/metabolismo , DNA Espaçador Ribossômico/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas Nucleares/metabolismo , Processamento Pós-Transcricional do RNA , Proteínas de Ligação a RNA/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/metabolismo
14.
Am J Hum Genet ; 100(3): 506-522, 2017 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-28257692

RESUMO

Ribosomal protein (RP) gene mutations, mostly associated with inherited or acquired bone marrow failure, are believed to drive disease by slowing the rate of protein synthesis. Here de novo missense mutations in the RPS23 gene, which codes for uS12, are reported in two unrelated individuals with microcephaly, hearing loss, and overlapping dysmorphic features. One individual additionally presents with intellectual disability and autism spectrum disorder. The amino acid substitutions lie in two highly conserved loop regions of uS12 with known roles in maintaining the accuracy of mRNA codon translation. Primary cells revealed one substitution severely impaired OGFOD1-dependent hydroxylation of a neighboring proline residue resulting in 40S ribosomal subunits that were blocked from polysome formation. The other disrupted a predicted pi-pi stacking interaction between two phenylalanine residues leading to a destabilized uS12 that was poorly tolerated in 40S subunit biogenesis. Despite no evidence of a reduction in the rate of mRNA translation, these uS12 variants impaired the accuracy of mRNA translation and rendered cells highly sensitive to oxidative stress. These discoveries describe a ribosomopathy linked to uS12 and reveal mechanistic distinctions between RP gene mutations driving hematopoietic disease and those resulting in developmental disorders.


Assuntos
Proteínas Ribossômicas/genética , Ribossomos/genética , Transtorno do Espectro Autista/genética , Proteínas de Transporte/genética , Células Cultivadas , Criança , Pré-Escolar , Códon/genética , Deficiências do Desenvolvimento/genética , Exoma , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Variação Genética , Perda Auditiva/genética , Humanos , Deficiência Intelectual/genética , Masculino , Microcefalia/genética , Mutação , Mutação de Sentido Incorreto , Proteínas Nucleares/genética , Estresse Oxidativo , Biossíntese de Proteínas/genética , Alinhamento de Sequência , Análise de Sequência de DNA
15.
Nucleic Acids Res ; 44(17): 8465-78, 2016 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-27530427

RESUMO

Synthesis of ribosomal subunits in eukaryotes is a complex and tightly regulated process that has been mostly characterized in yeast. The discovery of a growing number of diseases linked to defects in ribosome biogenesis calls for a deeper understanding of these mechanisms and of the specificities of human ribosome maturation. We present the 19 Å resolution cryo-EM reconstruction of a cytoplasmic precursor to the human small ribosomal subunit, purified by using the tagged ribosome biogenesis factor LTV1 as bait. Compared to yeast pre-40S particles, this first three-dimensional structure of a human 40S subunit precursor shows noticeable differences with respect to the position of ribosome biogenesis factors and uncovers the early deposition of the ribosomal protein RACK1 during subunit maturation. Consistently, RACK1 is required for efficient processing of the 18S rRNA 3'-end, which might be related to its role in translation initiation. This first structural analysis of a human pre-ribosomal particle sets the grounds for high-resolution studies of conformational transitions accompanying ribosomal subunit maturation.


Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Processamento Pós-Transcricional do RNA/genética , RNA Ribossômico 18S/genética , Receptores de Superfície Celular/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/química , Subunidades Ribossômicas Menores de Eucariotos/metabolismo , Sequência Conservada , Microscopia Crioeletrônica , Citoplasma/metabolismo , Células HEK293 , Células HeLa , Humanos , Modelos Moleculares , Biogênese de Organelas , Ligação Proteica , Receptores de Quinase C Ativada , Proteínas Ribossômicas/metabolismo , Subunidades Ribossômicas Menores de Eucariotos/ultraestrutura , Saccharomyces cerevisiae/metabolismo
16.
Wiley Interdiscip Rev RNA ; 6(2): 225-42, 2015 Mar-Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25346433

RESUMO

Ribosomal RNAs are the most abundant and universal noncoding RNAs in living organisms. In eukaryotes, three of the four ribosomal RNAs forming the 40S and 60S subunits are borne by a long polycistronic pre-ribosomal RNA. A complex sequence of processing steps is required to gradually release the mature RNAs from this precursor, concomitant with the assembly of the 79 ribosomal proteins. A large set of trans-acting factors chaperone this process, including small nucleolar ribonucleoparticles. While yeast has been the gold standard for studying the molecular basis of this process, recent technical advances have allowed to further define the mechanisms of ribosome biogenesis in animals and plants. This renewed interest for a long-lasting question has been fueled by the association of several genetic diseases with mutations in genes encoding both ribosomal proteins and ribosome biogenesis factors, and by the perspective of new anticancer treatments targeting the mechanisms of ribosome synthesis. A consensus scheme of pre-ribosomal RNA maturation is emerging from studies in various kinds of eukaryotic organisms. However, major differences between mammalian and yeast pre-ribosomal RNA processing have recently come to light.


Assuntos
Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA , Animais , Modelos Biológicos , Modelos Moleculares , Plantas
17.
Gastroenterology ; 147(3): 595-598.e5, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24941021

RESUMO

Little is known about the genetic factors that contribute to familial colorectal cancer type X (FCCX), characterized by hereditary nonpolyposis colorectal carcinoma with no mismatch repair defects. Genetic linkage analysis, exome sequencing, tumor studies, and functional investigations of 4 generations of a FCCX family led to the identification of a truncating germline mutation in RPS20, which encodes a component (S20) of the small ribosomal subunit and is a new colon cancer predisposition gene. The mutation was associated with a defect in pre-ribosomal RNA maturation. Our findings show that mutations in a gene encoding a ribosomal protein can predispose individuals to microsatellite-stable colon cancer. Evaluation of additional FCCX families for mutations in RPS20 and other ribosome-associated genes is warranted.


Assuntos
Neoplasias Colorretais Hereditárias sem Polipose/genética , Reparo de Erro de Pareamento de DNA , Mutação em Linhagem Germinativa , Proteínas Ribossômicas/genética , Análise Mutacional de DNA , Exossomos , Feminino , Ligação Genética , Predisposição Genética para Doença , Testes Genéticos/métodos , Hereditariedade , Humanos , Masculino , Pessoa de Meia-Idade , Linhagem , Fenótipo , Fatores de Risco
18.
Hum Genet ; 132(11): 1265-74, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-23812780

RESUMO

Diamond-Blackfan anemia (DBA) is an inherited red blood cell aplasia that usually presents during the first year of life. The main features of the disease are normochromic and macrocytic anemia, reticulocytopenia, and nearly absent erythroid progenitors in the bone marrow. The patients also present with growth retardation and craniofacial, upper limb, heart and urinary system congenital malformations in ~30-50 % of cases. The disease has been associated with point mutations and large deletions in ten ribosomal protein (RP) genes RPS19, RPS24, RPS17, RPL35A, RPL5, RPL11, RPS7, RPS10, RPS26, and RPL26 and GATA1 in about 60-65 % of patients. Here, we report a novel large deletion in RPL15, a gene not previously implicated to be causative in DBA. Like RPL26, RPL15 presents the distinctive feature of being required both for 60S subunit formation and for efficient cleavage of the internal transcribed spacer 1. In addition, we detected five deletions in RP genes in which mutations have been previously shown to cause DBA: one each in RPS19, RPS24, and RPS26, and two in RPS17. Pre-ribosomal RNA processing was affected in cells established from the patients bearing these deletions, suggesting a possible molecular basis for their pathological effect. These data identify RPL15 as a new gene involved in DBA and further support the presence of large deletions in RP genes in DBA patients.


Assuntos
Anemia de Diamond-Blackfan/genética , Deleção de Genes , Proteínas Ribossômicas/genética , Hibridização Genômica Comparativa , Variações do Número de Cópias de DNA , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Mutação , RNA Ribossômico/análise , RNA Ribossômico/genética , RNA Interferente Pequeno , Proteínas Ribossômicas/metabolismo
19.
Nucleic Acids Res ; 41(8): 4709-23, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23482395

RESUMO

Defects in ribosome biogenesis trigger stress response pathways, which perturb cell proliferation and differentiation in several genetic diseases. In Diamond-Blackfan anemia (DBA), a congenital erythroblastopenia, mutations in ribosomal protein genes often interfere with the processing of the internal transcribed spacer 1 (ITS1), the mechanism of which remains elusive in human cells. Using loss-of-function experiments and extensive RNA analysis, we have defined the precise position of the endonucleolytic cleavage E in the ITS1, which generates the 18S-E intermediate, the last precursor to the 18S rRNA. Unexpectedly, this cleavage is followed by 3'-5' exonucleolytic trimming of the 18S-E precursor during nuclear export of the pre-40S particle, which sets a new mechanism for 18S rRNA formation clearly different from that established in yeast. In addition, cleavage at site E is also followed by 5'-3' exonucleolytic trimming of the ITS1 by exonuclease XRN2. Perturbation of this step on knockdown of the large subunit ribosomal protein RPL26, which was recently associated to DBA, reveals the putative role of a highly conserved cis-acting sequence in ITS1 processing. These data cast new light on the original mechanism of ITS1 elimination in human cells and provide a mechanistic framework to further study the interplay of DBA-linked ribosomal proteins in this process.


Assuntos
Nucléolo Celular/enzimologia , Citoplasma/enzimologia , Processamento Pós-Transcricional do RNA , RNA Ribossômico 18S/metabolismo , Sequência de Bases , Sequência Conservada , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Células HeLa , Humanos , Precursores de RNA/metabolismo , RNA Ribossômico 18S/biossíntese , RNA Ribossômico 18S/química , Proteínas Ribossômicas/metabolismo
20.
J Cell Sci ; 125(Pt 19): 4532-42, 2012 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-22767511

RESUMO

Understanding the relationship between the topological dynamics of nuclear subdomains and their molecular function is a central issue in nucleus biology. Pre-nucleolar bodies (PNBs) are transient nuclear subdomains, which form at telophase and contain nucleolar proteins, snoRNPs and pre-ribosomal RNAs (pre-rRNAs). These structures gradually disappear in early G1 phase and are currently regarded as reservoirs of nucleolar factors that participate to post-mitotic reassembly of the nucleolus. Here, we provide evidence from fluorescence in situ hybridization and loss-of-function experiments in HeLa cells that PNBs are in fact active ribosome factories in which maturation of the pre-rRNAs transiting through mitosis resumes at telophase. We show that the pre-rRNA spacers are sequentially removed in PNBs when cells enter G1 phase, indicating regular pre-rRNA processing as in the nucleolus. Accordingly, blocking pre-rRNA maturation induces accumulation in PNBs of stalled pre-ribosomes characterised by specific pre-rRNAs and pre-ribosomal factors. The presence of pre-ribosomal particles in PNBs is corroborated by observation of these domains by correlative electron tomography. Most importantly, blocking pre-rRNA maturation also prevents the gradual disappearance of PNBs, which persist for several hours in the nucleoplasm. In a revised model, we propose that PNBs are autonomous extra-nucleolar ribosome maturation sites, whose orderly disassembly in G1 phase is driven by the maturation and release of their pre-ribosome content.


Assuntos
Nucléolo Celular/metabolismo , Mitose/genética , Precursores de RNA/metabolismo , Processamento Pós-Transcricional do RNA/genética , Nucléolo Celular/ultraestrutura , Tomografia com Microscopia Eletrônica , Fase G1/genética , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , Modelos Biológicos , Precursores de RNA/genética , RNA Interferente Pequeno/metabolismo , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , Proteínas Ribossômicas/metabolismo
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