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1.
Sci Rep ; 9(1): 895, 2019 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-30696890

RESUMO

The prognosis of patients with relapsed/refractory (R/R) diffuse large B-cell lymphoma (DLBCL) remains unsatisfactory and, despite major advances in genomic studies, the biological mechanisms underlying chemoresistance are still poorly understood. We conducted for the first time a large-scale differential multi-omics investigation on DLBCL patient's samples in order to identify new biomarkers that could early identify patients at risk of R/R disease and to identify new targets that could determine chemorefractoriness. We compared a well-characterized cohort of R/R versus chemosensitive DLBCL patients by combining label-free quantitative proteomics and targeted RNA sequencing performed on the same tissues samples. The cross-section of both data levels allowed extracting a sub-list of 22 transcripts/proteins pairs whose expression levels significantly differed between the two groups of patients. In particular, we identified significant targets related to tumor metabolism (Hexokinase 3), microenvironment (IDO1, CXCL13), cancer cells proliferation, migration and invasion (S100 proteins) or BCR signaling pathway (CD79B). Overall, this study revealed several extremely promising biomarker candidates related to DLBCL chemorefractoriness and highlighted some new potential therapeutic drug targets. The complete datasets have been made publically available and should constitute a valuable resource for the future research.

2.
Am J Hum Genet ; 104(2): 319-330, 2019 02 07.
Artigo em Inglês | MEDLINE | ID: mdl-30639322

RESUMO

ZMIZ1 is a coactivator of several transcription factors, including p53, the androgen receptor, and NOTCH1. Here, we report 19 subjects with intellectual disability and developmental delay carrying variants in ZMIZ1. The associated features include growth failure, feeding difficulties, microcephaly, facial dysmorphism, and various other congenital malformations. Of these 19, 14 unrelated subjects carried de novo heterozygous single-nucleotide variants (SNVs) or single-base insertions/deletions, 3 siblings harbored a heterozygous single-base insertion, and 2 subjects had a balanced translocation disrupting ZMIZ1 or involving a regulatory region of ZMIZ1. In total, we identified 13 point mutations that affect key protein regions, including a SUMO acceptor site, a central disordered alanine-rich motif, a proline-rich domain, and a transactivation domain. All identified variants were absent from all available exome and genome databases. In vitro, ZMIZ1 showed impaired coactivation of the androgen receptor. In vivo, overexpression of ZMIZ1 mutant alleles in developing mouse brains using in utero electroporation resulted in abnormal pyramidal neuron morphology, polarization, and positioning, underscoring the importance of ZMIZ1 in neural development and supporting mutations in ZMIZ1 as the cause of a rare neurodevelopmental syndrome.


Assuntos
Deficiências do Desenvolvimento/genética , Deficiência Intelectual/genética , Mutação Puntual , Fatores de Transcrição/genética , Alelos , Animais , Criança , Pré-Escolar , Deficiências do Desenvolvimento/patologia , Feminino , Humanos , Lactente , Deficiência Intelectual/patologia , Masculino , Camundongos , Síndrome , Fatores de Transcrição/química , Fatores de Transcrição/metabolismo
3.
PLoS Pathog ; 14(10): e1007368, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30335851

RESUMO

Infection with human BK polyomavirus, a small double-stranded DNA virus, potentially results in severe complications in immunocompromised patients. Here, we describe the in vivo variability and evolution of the BK polyomavirus by deep sequencing. Our data reveal the highest genomic evolutionary rate described in double-stranded DNA viruses, i.e., 10-3-10-5 substitutions per nucleotide site per year. High mutation rates in viruses allow their escape from immune surveillance and adaptation to new hosts. By combining mutational landscapes across viral genomes with in silico prediction of viral peptides, we demonstrate the presence of significantly more coding substitutions within predicted cognate HLA-C-bound viral peptides than outside. This finding suggests a role for HLA-C in antiviral immunity, perhaps through the action of killer cell immunoglobulin-like receptors. The present study provides a comprehensive view of viral evolution and immune escape in a DNA virus.

4.
Ann Rheum Dis ; 77(11): 1675-1687, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30030262

RESUMO

OBJECTIVES: The objective of the present study was to explain why two siblings carrying both the same homozygous pathogenic mutation for the autoinflammatory disease hyper IgD syndrome, show opposite phenotypes, that is, the first being asymptomatic, the second presenting all classical characteristics of the disease. METHODS: Where single omics (mainly exome) analysis fails to identify culprit genes/mutations in human complex diseases, multiomics analyses may provide solutions, although this has been seldom used in a clinical setting. Here we combine exome, transcriptome and proteome analyses to decipher at a molecular level, the phenotypic differences between the two siblings. RESULTS: This multiomics approach led to the identification of a single gene-STAT1-which harboured a rare missense variant and showed a significant overexpression of both mRNA and protein in the symptomatic versus the asymptomatic sister. This variant was shown to be of gain of function nature, involved in an increased activation of the Janus kinase/signal transducer and activator of transcription signalling (JAK/STAT) pathway, known to play a critical role in inflammatory diseases and for which specific biotherapies presently exist. Pathway analyses based on information from differentially expressed transcripts and proteins confirmed the central role of STAT1 in the proposed regulatory network leading to an increased inflammatory phenotype in the symptomatic sibling. CONCLUSIONS: This study demonstrates the power of a multiomics approach to uncover potential clinically actionable targets for a personalised therapy. In more general terms, we provide a proteogenomics analysis pipeline that takes advantage of subject-specific genomic and transcriptomic information to improve protein identification and hence advance individualised medicine.

5.
Dev Cell ; 45(1): 33-52.e12, 2018 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-29634935

RESUMO

Metastatic seeding is driven by cell-intrinsic and environmental cues, yet the contribution of biomechanics is poorly known. We aim to elucidate the impact of blood flow on the arrest and the extravasation of circulating tumor cells (CTCs) in vivo. Using the zebrafish embryo, we show that arrest of CTCs occurs in vessels with favorable flow profiles where flow forces control the adhesion efficacy of CTCs to the endothelium. We biophysically identified the threshold values of flow and adhesion forces allowing successful arrest of CTCs. In addition, flow forces fine-tune tumor cell extravasation by impairing the remodeling properties of the endothelium. Importantly, we also observe endothelial remodeling at arrest sites of CTCs in mouse brain capillaries. Finally, we observed that human supratentorial brain metastases preferably develop in areas with low perfusion. These results demonstrate that hemodynamic profiles at metastatic sites regulate key steps of extravasation preceding metastatic outgrowth.


Assuntos
Neoplasias Encefálicas/secundário , Neoplasias da Mama/patologia , Adesão Celular , Hemodinâmica , Neoplasias Pulmonares/patologia , Melanoma/patologia , Células Neoplásicas Circulantes/patologia , Animais , Neoplasias Encefálicas/metabolismo , Neoplasias da Mama/metabolismo , Ciclo Celular , Circulação Cerebrovascular , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Endotélio Vascular/citologia , Endotélio Vascular/metabolismo , Feminino , Humanos , Neoplasias Pulmonares/metabolismo , Masculino , Melanoma/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células Neoplásicas Circulantes/metabolismo , Estudos Retrospectivos , Células Tumorais Cultivadas , Peixe-Zebra
6.
J Clin Invest ; 127(11): 4090-4103, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28972538

RESUMO

Shwachman-Diamond syndrome (SDS) (OMIM #260400) is a rare inherited bone marrow failure syndrome (IBMFS) that is primarily characterized by neutropenia and exocrine pancreatic insufficiency. Seventy-five to ninety percent of patients have compound heterozygous loss-of-function mutations in the Shwachman-Bodian-Diamond syndrome (sbds) gene. Using trio whole-exome sequencing (WES) in an sbds-negative SDS family and candidate gene sequencing in additional SBDS-negative SDS cases or molecularly undiagnosed IBMFS cases, we identified 3 independent patients, each of whom carried a de novo missense variant in srp54 (encoding signal recognition particle 54 kDa). These 3 patients shared congenital neutropenia linked with various other SDS phenotypes. 3D protein modeling revealed that the 3 variants affect highly conserved amino acids within the GTPase domain of the protein that are critical for GTP and receptor binding. Indeed, we observed that the GTPase activity of the mutated proteins was impaired. The level of SRP54 mRNA in the bone marrow was 3.6-fold lower in patients with SRP54-mutations than in healthy controls. Profound reductions in neutrophil counts and chemotaxis as well as a diminished exocrine pancreas size in a SRP54-knockdown zebrafish model faithfully recapitulated the human phenotype. In conclusion, autosomal dominant mutations in SRP54, a key member of the cotranslation protein-targeting pathway, lead to syndromic neutropenia with a Shwachman-Diamond-like phenotype.


Assuntos
Doenças da Medula Óssea/genética , Insuficiência Pancreática Exócrina/genética , Lipomatose/genética , Neutropenia/congênito , Partícula de Reconhecimento de Sinal/genética , Animais , Criança , Análise Mutacional de DNA , Feminino , Estudos de Associação Genética , Humanos , Lactente , Masculino , Modelos Moleculares , Neutropenia/genética , Pâncreas Exócrino/metabolismo , Fenótipo , Domínios Proteicos , Partícula de Reconhecimento de Sinal/química , Peixe-Zebra
7.
EBioMedicine ; 21: 158-169, 2017 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-28615147

RESUMO

HIV-specific broadly neutralizing antibodies (bnAbs) have been isolated from patients with high viremia but also from HIV controllers that repress HIV-1 replication. In these elite controllers (ECs), multiple parameters contribute to viral suppression, including genetic factors and immune responses. Defining the immune correlates associated with the generation of bnAbs may help in designing efficient immunotherapies. In this study, in ECs either positive or negative for the HLA-B*57 protective allele, in treated HIV-infected and HIV-negative individuals, we characterized memory B cell compartments and HIV-specific memory B cells responses using flow cytometry and ELISPOT. ECs preserved their memory B cell compartments and in contrast to treated patients, maintained detectable HIV-specific memory B cell responses. All ECs presented IgG1+ HIV-specific memory B cells but some individuals also preserved IgG2+ or IgG3+ responses. Importantly, we also analyzed the capacity of sera from ECs to neutralize a panel of HIV strains including transmitted/founder virus. 29% and 21% of HLA-B*57+ and HLA-B*57- ECs, respectively, neutralized at least 40% of the viral strains tested. Remarkably, in HLA-B*57+ ECs the frequency of HIV-Env-specific memory B cells correlated positively with the neutralization breadth suggesting that preservation of HIV-specific memory B cells might contribute to the neutralizing responses in these patients.


Assuntos
Anticorpos Neutralizantes/imunologia , Linfócitos B/imunologia , Anticorpos Anti-HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Contagem de Linfócitos , Linfócitos B/metabolismo , ELISPOT , Feminino , Infecções por HIV/epidemiologia , Infecções por HIV/virologia , Antígenos HLA-B/imunologia , Humanos , Imunoglobulina G/sangue , Imunoglobulina G/imunologia , Memória Imunológica , Masculino , Testes de Neutralização , Carga Viral
8.
Genome Biol Evol ; 9(5)2017 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-28444231

RESUMO

Coagulase negative staphylococci are normal inhabitant of the human skin flora that account for an increasing number of infections, particularly hospital-acquired infections. Staphylococcus lugdunensis has emerged as a most virulent species causing various infections with clinical characteristics close to what clinicians usually observe with Staphylococcus aureus and both bacteria share more than 70% of their genome. Virulence of S. aureus relies on a large repertoire of virulence factors, many of which are encoded on mobile genetic elements. S. lugdunensis also bears various putative virulence genes but only one complete genome with extensive analysis has been published with one prophage sequence (φSL2) and a unique plasmid was previously described. In this study, we performed de novo sequencing, whole genome assembly and annotation of seven strains of S. lugdunensis from VISLISI clinical trial. We searched for the presence of virulence genes and mobile genetics elements using bioinformatics tools. We identified four new prophages, named φSL2 to φSL4, belonging to the Siphoviridae class and five plasmids, named pVISLISI_1 to pVISLISI_5. Three plasmids are homologous to known plasmids that include, amongst others, one S. aureus plasmid. The two other plasmids were not described previously. This study provides a new context for the study of S. lugdunensis virulence suggesting the occurrence of several genetic recombination' with other staphylococci.


Assuntos
Staphylococcus lugdunensis/classificação , Staphylococcus lugdunensis/genética , Genoma Bacteriano , Ilhas Genômicas , Sequências Repetitivas Dispersas , Anotação de Sequência Molecular , Prófagos , Recombinação Genética , Infecções Estafilocócicas/microbiologia , Staphylococcus lugdunensis/patogenicidade , Fatores de Virulência/genética
9.
Hum Mol Genet ; 26(13): 2565-2576, 2017 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-28379387

RESUMO

The association of primary Sjögren's syndrome (pSS) with Major Histocompatibility Complex (MHC) alleles is quintessential of MHC-disease associations. Indeed, although disease associations with classical HLA class I and II alleles/haplotypes are amply documented, further dissection is often prevented by the strong linkage disequilibrium across the entire MHC complex. Here we study the association of pSS, not with HLA genes, but with the non-conventional MHC encoded class I gene, MICA (MHC class I chain-related gene A). MICA is selectively expressed within epithelia, and is the major ligand for the activatory receptor, NKG2D, both attributes relevant to pSS' etiology. MICA-pSS association was studied in two independent (French and UK) cohorts representing a total of 959 cases and 1,043 controls. MICA*008 allele was shown to be significantly associated with pSS (pcor=2.61 × 10-35). A multivariate logistic regression showed that this association was independent of all major known MHC-linked risk loci/alleles, as well as other relevant candidate loci that are in linkage disequilibrium with MICA*008 i.e. HLA-B*08:01, rs3131619 (T), MICB*008, TNF308A, HLA-DRB1*03:01 and HLA-DRB1*15:01 (P = 1.84 × 10-04). Furthermore, independently of the MICA*008 allele, higher levels of soluble MICA proteins were detected in sera of pSS patients compared to healthy controls. This study hence defines MICA as a new, MHC-linked, yet HLA-independent, pSS risk locus and opens a new front in our understanding of the still enigmatic pathophysiology of this disease. The fact that the soluble MICA protein is further amplified in MICA*008 carrying individuals, might also be relevant in other auto-immune diseases and cancer.


Assuntos
Antígenos de Histocompatibilidade Classe I/genética , Síndrome de Sjogren/genética , Adulto , Alelos , Grupo com Ancestrais do Continente Europeu/genética , Feminino , Frequência do Gene/genética , Predisposição Genética para Doença , Antígenos HLA-B/genética , Cadeias HLA-DRB1/genética , Haplótipos , Antígenos de Histocompatibilidade Classe I/metabolismo , Humanos , Desequilíbrio de Ligação , Complexo Principal de Histocompatibilidade/genética , Masculino , Pessoa de Meia-Idade , Polimorfismo Genético
10.
Eur J Hum Genet ; 24(12): 1746-1751, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27381093

RESUMO

Spondylocarpotarsal synostosis syndrome (SCT) is a rare Mendelian disorder (OMIM #272460) characterized by prenatal vertebral fusion, scoliosis, short stature and carpal and tarsal synostosis. SCT is typically known as an autosomal recessive disease caused by variants in the FLNB gene. The genetic basis of the rarer cases of vertical transmissions remains unknown. In two independent families with symptoms related to autosomal dominant SCT, we identified - by exome sequencing - two protein-altering variants in the embryonic myosin heavy chain 3 (MYH3) gene. As MYH3 variants are also associated with distal arthrogryposis (DA1, DA2A, DA2B) and autosomal dominant multiple pterygium syndromes (MPS), the present study expands the phenotypic spectrum of MYH3 variants to autosomal dominant SCT. Vertebral, carpal and tarsal fusions observed in both families further confirm that MYH3 plays a key role in skeletal development.


Assuntos
Anormalidades Múltiplas/genética , Proteínas do Citoesqueleto/genética , Vértebras Lombares/anormalidades , Doenças Musculoesqueléticas/genética , Mutação , Fenótipo , Escoliose/congênito , Sinostose/genética , Vértebras Torácicas/anormalidades , Anormalidades Múltiplas/patologia , Adolescente , Adulto , Criança , Exoma , Feminino , Humanos , Vértebras Lombares/patologia , Masculino , Doenças Musculoesqueléticas/patologia , Linhagem , Escoliose/genética , Escoliose/patologia , Sinostose/patologia , Vértebras Torácicas/patologia
11.
Arthritis Rheumatol ; 68(8): 1839-48, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26882526

RESUMO

OBJECTIVE: While the regulatory role of individual microRNAs (miRNAs) in rheumatoid arthritis (RA) is well established, the role of DICER1 in the pathogenesis of the disease has not yet been investigated. The purpose of this study was to analyze the expression of factors involved in miRNA biogenesis in fibroblast-like synoviocytes (FLS) from RA patients and to monitor the arthritis triggered by K/BxN serum transfer in mice deficient in the Dicer gene (Dicer(d/d) ). METHODS: The expression of genes and precursor miRNAs was quantified by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). MicroRNA macroarray profiling was monitored by qRT-PCR. Cytokines were quantified by enzyme-linked immunosorbent assay. Experimental arthritis in mice was achieved by the transfer of serum from K/BxN donors. Apoptosis was quantified using an enzyme-linked immunosorbent assay. RESULTS: We found decreased DICER1 and mature miRNA expression in synovial fibroblasts from RA patients. These cells were hyperresponsive to lipopolysaccharide, as evidenced by their increased interleukin-6 secretion upon stimulation. Experimental serum-transfer arthritis in Dicer(d/d) mice confirmed that an unbalanced biogenesis of miRNAs correlated with an enhanced inflammatory response. Synoviocytes from both RA patients and Dicer(d/d) mice exhibited increased resistance to apoptotic stimuli. CONCLUSION: The findings of this study further substantiate the important role of DICER1 in the maintenance of homeostasis and the regulation of inflammatory responses.


Assuntos
Artrite Reumatoide/genética , RNA Helicases DEAD-box/genética , Ribonuclease III/genética , Sinoviócitos/fisiologia , Animais , Apoptose , Regulação da Expressão Gênica , Humanos , Inflamação/genética , Camundongos
12.
Dev Cell ; 36(1): 63-78, 2016 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-26766443

RESUMO

Mitosis ensures equal segregation of the genome and is controlled by a variety of ubiquitylation signals on substrate proteins. However, it remains unexplored how the versatile ubiquitin code is read out during mitotic progression. Here, we identify the ubiquitin receptor protein UBASH3B as an important regulator of mitosis. UBASH3B interacts with ubiquitylated Aurora B, one of the main kinases regulating chromosome segregation, and controls its subcellular localization but not protein levels. UBASH3B is a limiting factor in this pathway and is sufficient to localize Aurora B to microtubules prior to anaphase. Importantly, targeting Aurora B to microtubules by UBASH3B is necessary for the timing and fidelity of chromosome segregation in human cells. Our findings uncover an important mechanism defining how ubiquitin attachment to a substrate protein is decoded during mitosis.


Assuntos
Aurora Quinase B/metabolismo , Segregação de Cromossomos/genética , Microtúbulos/metabolismo , Mitose/fisiologia , Proteínas Tirosina Fosfatases/metabolismo , Ubiquitina/metabolismo , Anáfase/fisiologia , Linhagem Celular , Células HeLa , Humanos , Cinetocoros/metabolismo , Fosforilação , Ubiquitinação/fisiologia
13.
PLoS One ; 10(11): e0141740, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26524763

RESUMO

Eosinophils are leukocytes that are released into the peripheral blood in a phenotypically mature state and are capable of being recruited into tissues in response to appropriate stimuli. Eosinophils, traditionally considered cytotoxic effector cells, are leukocytes recruited into the airways of asthma patients where they are believed to contribute to the development of many features of the disease. This perception, however, has been challenged by recent findings suggesting that eosinophils have also immunomodulatory functions and may be involved in tissue homeostasis and wound healing. Here we describe a transcriptome-based approach-in a limited number of patients and controls-to investigate the activation state of circulating human eosinophils isolated by flow cytometry. We provide an overview of the global expression pattern in eosinophils in various relevant conditions, e.g., eosinophilic asthma, hypereosinophilic dermatological diseases, parasitosis and pulmonary aspergillosis. Compared to healthy subjects, circulating eosinophils isolated from asthma patients differed in their gene expression profile which is marked by downregulation of transcripts involved in antigen presentation, pathogen recognition and mucosal innate immunity, whereas up-regulated genes were involved in response to non-specific stimulation, wounding and maintenance of homeostasis. Eosinophils from other hypereosinophilic disorders displayed a very similar transcriptional profile. Taken together, these observations seem to indicate that eosinophils exhibit non-specific immunomodulatory functions important for tissue repair and homeostasis and suggest new roles for these cells in asthma immunobiology.


Assuntos
Asma/genética , Eosinófilos/citologia , Síndrome Hipereosinofílica/genética , Transcriptoma , Adulto , Idoso , Asma/sangue , Eosinófilos/metabolismo , Feminino , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica , Humanos , Síndrome Hipereosinofílica/sangue , Masculino , Pessoa de Meia-Idade
14.
Mov Disord ; 30(3): 423-7, 2015 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-25545163

RESUMO

IMPORTANCE: Apart from Huntington's disease, little is known of the genetics of autosomal dominant chorea associated with dystonia. Here we identify adenylate cyclase 5 (ADCY5) as a likely new causal gene for early-onset chorea and dystonia. OBSERVATIONS: Whole exome sequencing in a three-generation family affected with autosomal dominant chorea associated with dystonia identified a single de novo mutation­c.2088+1G>A in a 5' donor splice-site of ADCY5­segregating with the disease. This mutation seeming leads to RNA instability and therefore ADCY5 haploinsufficiency. CONCLUSIONS AND RELEVANCE: Our finding confirms the genetic/clinical heterogeneity of the disorder; corroborated by previous identification of ADCY5 mutations in one family with dyskinesia-facial myokymia and in two unrelated sporadic cases of paxoysmal choreic/dystonia-facial myokymia; ADCY5's high expression in the striatum and movement disorders in ADCY5-deficient mice. Hence ADCY5 genetic analyses may be relevant in the diagnostic workup of unexplained early-onset hyperkinetic movement disorders.


Assuntos
Adenilil Ciclases/genética , Coreia/genética , Distúrbios Distônicos/genética , Saúde da Família , Mutação/genética , Adolescente , Adulto , Análise Mutacional de DNA , Feminino , Humanos , Masculino
15.
J Hum Genet ; 59(1): 57-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24225993

RESUMO

Noonan syndrome (NS), an autosomal dominant multisystem disorder, is caused by the dysregulation of the RAS-MAPK pathway and is characterized by short stature, heart defects, pectus excavatum, webbed neck, learning problems, cryptorchidism and facial dysmorphism. We here present the clinical and molecular characterization of a family with NS and multiple giant cell lesions (MGCLs). The proband is a 12-year-old girl with NS and MGCL. Her mother shows typical NS without MGCL. Whole-exome sequencing of the girl, her mother and her healthy maternal grand parents revealed a previously unobserved mutation in exon 5 of the PTPN11 gene (c.598 A>T; p.N200Y), transmitted from the mother to the proband. As no other modification in the RAS-MAPK pathway genes as related to Rasopathies was detected in the proband, this report demonstrates for the first time that a unique mutation affecting this, otherwise unaffected signaling route, can cause both NS and NS/MGCL in the same family. This observation further confirms that NS/MGCL is not a distinct entity but rather that MGCL represents a rare complication of NS. Moreover, the localization of the p.N200Y mutation suggests an alternative molecular mechanism for the excessive phosphatase activity of the PTPN11-encoded protein.


Assuntos
Células Gigantes/patologia , Mutação , Síndrome de Noonan/genética , Síndrome de Noonan/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 11/genética , Domínios de Homologia de src/genética , Biópsia , Criança , Análise Mutacional de DNA , Exoma , Facies , Feminino , Humanos , Masculino , Fenótipo , Proteína Tirosina Fosfatase não Receptora Tipo 11/química , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia
16.
Proc Natl Acad Sci U S A ; 108(51): 20603-8, 2011 Dec 20.
Artigo em Inglês | MEDLINE | ID: mdl-22147914

RESUMO

SRC-3 is an important coactivator of nuclear receptors including the retinoic acid (RA) receptor α. Most of SRC-3 functions are facilitated by changes in the posttranslational code of the protein that involves mainly phosphorylation and ubiquitination. We recently reported that SRC-3 is degraded by the proteasome in response to RA. Here, by using an RNAi E3-ubiquitin ligase entry screen, we identified CUL-3 and RBX1 as components of the E3 ubiquitin ligase involved in the RA-induced ubiquitination and subsequent degradation of SRC-3. We also show that the RA-induced ubiquitination of SRC-3 depends on its prior phosphorylation at serine 860 that promotes binding of the CUL-3-based E3 ligase in the nucleus. Finally, phosphorylation, ubiquitination, and degradation of SRC-3 cooperate to control the dynamics of transcription. In all, this process participates to the antiproliferative effect of RA.


Assuntos
Proteínas Culina/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Tretinoína/química , Ubiquitina/química , Animais , Sítios de Ligação , Células COS , Diferenciação Celular , Linhagem Celular Tumoral , Núcleo Celular/metabolismo , Proliferação de Células , Cercopithecus aethiops , Cromatina/química , Humanos , Microscopia de Fluorescência/métodos , Fosforilação , Transcrição Genética
17.
BMC Genomics ; 11: 565, 2010 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-20946641

RESUMO

BACKGROUND: In addition to acting as an RNA quality control pathway, nonsense-mediated mRNA decay (NMD) plays roles in regulating normal gene expression. In particular, the extent to which alternative splicing is coupled to NMD and the roles of NMD in regulating uORF containing transcripts have been a matter of debate. RESULTS: In order to achieve a greater understanding of NMD regulated gene expression we used 2D-DiGE proteomics technology to examine the changes in protein expression induced in HeLa cells by UPF1 knockdown. QPCR based validation of the corresponding mRNAs, in response to both UPF1 knockdown and cycloheximide treatment, identified 17 bona fide NMD targets. Most of these were associated with bioinformatically predicted NMD activating features, predominantly upstream open reading frames (uORFs). Strikingly, however, the majority of transcripts up-regulated by UPF1 knockdown were either insensitive to, or even down-regulated by, cycloheximide treatment. Furthermore, the mRNA abundance of several down-regulated proteins failed to change upon UPF1 knockdown, indicating that UPF1's role in regulating mRNA and protein abundance is more complex than previously appreciated. Among the bona fide NMD targets, we identified a highly conserved AS-NMD event within the 3' UTR of the HNRNPA2B1 gene. Overexpression of GFP tagged hnRNP A2 resulted in a decrease in endogenous hnRNP A2 and B1 mRNA with a concurrent increase in the NMD sensitive isoforms. CONCLUSIONS: Despite the large number of changes in protein expression upon UPF1 knockdown, a relatively small fraction of them can be directly attributed to the action of NMD on the corresponding mRNA. From amongst these we have identified a conserved AS-NMD event within HNRNPA2B1 that appears to mediate autoregulation of HNRNPA2B1 expression levels.


Assuntos
Processamento Alternativo/genética , Códon sem Sentido/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Homeostase/genética , Proteômica/métodos , Estabilidade de RNA/genética , Transativadores/metabolismo , Regiões 3' não Traduzidas/genética , Proteínas de Transporte/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Ribonucleoproteína Nuclear Heterogênea A1 , Humanos , Espectrometria de Massas , Fases de Leitura Aberta/genética , Fosfatidilinositol 3-Quinases/metabolismo , Biossíntese de Proteínas/genética , RNA Helicases , RNA Antissenso/metabolismo , Reprodutibilidade dos Testes , Fatores de Transcrição/metabolismo , Eletroforese em Gel Diferencial Bidimensional
18.
DNA Res ; 15(2): 63-72, 2008 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-18276623

RESUMO

High-throughput sequencing studies revealed that the majority of human and mouse multi-exon genes have multiple splice forms. High-density oligonucleotide array-based measurements have further established that many exons are expressed in a tissue-specific manner. The mechanisms underlying the tissue-dependent expression of most alternative exons remain, however, to be understood. In this study, we focus on one possible mechanism, namely the coupling of (tissue specific) transcription regulation with alternative splicing. We analyzed the FANTOM3 and H-Invitational datasets of full-length mouse and human cDNAs, respectively, and found that in transcription units with multiple start sites, the inclusion of at least 15% and possibly up to 30% of the 'cassette' exons correlates with the use of specific transcription start sites (TSS). The vast majority of TSS-associated exons are conserved between human and mouse, yet the conservation is weaker when compared with TSS-independent exons. Additionally, the currently available data only support a weak correlation between the probabilities of TSS association of orthologous exons. Our analysis thus suggests frequent coupling of transcriptional and splicing programs, and provides a large dataset of exons on which the molecular basis of this coupling can be further studied.


Assuntos
Processamento Alternativo , Biologia Computacional/métodos , DNA Complementar/genética , Regulação da Expressão Gênica , Transcrição Genética , Animais , Éxons/genética , Humanos , Camundongos , RNA Mensageiro/genética , Transdução de Sinais
19.
PLoS Genet ; 2(4): e24, 2006 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-16683023

RESUMO

Recent large-scale cDNA sequencing efforts show that elaborate patterns of splice variation are responsible for much of the proteome diversity in higher eukaryotes. To obtain an accurate account of the repertoire of splice variants, and to gain insight into the mechanisms of alternative splicing, it is essential that cDNAs are very accurately mapped to their respective genomes. Currently available algorithms for cDNA-to-genome alignment do not reach the necessary level of accuracy because they use ad hoc scoring models that cannot correctly trade off the likelihoods of various sequencing errors against the probabilities of different gene structures. Here we develop a Bayesian probabilistic approach to cDNA-to-genome alignment. Gene structures are assigned prior probabilities based on the lengths of their introns and exons, and based on the sequences at their splice boundaries. A likelihood model for sequencing errors takes into account the rates at which misincorporation, as well as insertions and deletions of different lengths, occurs during sequencing. The parameters of both the prior and likelihood model can be automatically estimated from a set of cDNAs, thus enabling our method to adapt itself to different organisms and experimental procedures. We implemented our method in a fast cDNA-to-genome alignment program, SPA, and applied it to the FANTOM3 dataset of over 100,000 full-length mouse cDNAs and a dataset of over 20,000 full-length human cDNAs. Comparison with the results of four other mapping programs shows that SPA produces alignments of significantly higher quality. In particular, the quality of the SPA alignments near splice boundaries and SPA's mapping of the 5' and 3' ends of the cDNAs are highly improved, allowing for more accurate identification of transcript starts and ends, and accurate identification of subtle splice variations. Finally, our splice boundary analysis on the human dataset suggests the existence of a novel non-canonical splice site that we also find in the mouse dataset. The SPA software package is available at http://www.biozentrum.unibas.ch/personal/nimwegen/cgi-bin/spa.cgi.


Assuntos
Processamento Alternativo , Algoritmos , Teorema de Bayes , Mapeamento Cromossômico , DNA Complementar/genética , Éxons , Modelos Genéticos , Mutação , Probabilidade , Sítios de Splice de RNA , Processamento de RNA/genética , Alinhamento de Sequência/métodos , Análise de Sequência de RNA , Deleção de Sequência , Transcrição Genética
20.
BMC Bioinformatics ; 6: 267, 2005 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-16274478

RESUMO

BACKGROUND: MicroRNAs (miRNAs) are endogenous 21 to 23-nucleotide RNA molecules that regulate protein-coding gene expression in plants and animals via the RNA interference pathway. Hundreds of them have been identified in the last five years and very recent works indicate that their total number is still larger. Therefore miRNAs gene discovery remains an important aspect of understanding this new and still widely unknown regulation mechanism. Bioinformatics approaches have proved to be very useful toward this goal by guiding the experimental investigations. RESULTS: In this work we describe our computational method for miRNA prediction and the results of its application to the discovery of novel mammalian miRNAs. We focus on genomic regions around already known miRNAs, in order to exploit the property that miRNAs are occasionally found in clusters. Starting with the known human, mouse and rat miRNAs we analyze 20 kb of flanking genomic regions for the presence of putative precursor miRNAs (pre-miRNAs). Each genome is analyzed separately, allowing us to study the species-specific identity and genome organization of miRNA loci. We only use cross-species comparisons to make conservative estimates of the number of novel miRNAs. Our ab initio method predicts between fifty and hundred novel pre-miRNAs for each of the considered species. Around 30% of these already have experimental support in a large set of cloned mammalian small RNAs. The validation rate among predicted cases that are conserved in at least one other species is higher, about 60%, and many of them have not been detected by prediction methods that used cross-species comparisons. A large fraction of the experimentally confirmed predictions correspond to an imprinted locus residing on chromosome 14 in human, 12 in mouse and 6 in rat. Our computational tool can be accessed on the world-wide-web. CONCLUSION: Our results show that the assumption that many miRNAs occur in clusters is fruitful for the discovery of novel miRNAs. Additionally we show that although the overall miRNA content in the observed clusters is very similar across the three considered species, the internal organization of the clusters changes in evolution.


Assuntos
Biologia Computacional/métodos , MicroRNAs/química , Algoritmos , Animais , Mapeamento Cromossômico , Análise por Conglomerados , Genoma , Humanos , Internet , Camundongos , Modelos Genéticos , Família Multigênica , Conformação de Ácido Nucleico , RNA/química , Ratos , Sensibilidade e Especificidade , Alinhamento de Sequência , Software
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