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1.
Vestn Oftalmol ; 135(5. Vyp. 2): 192-198, 2019.
Artigo em Russo | MEDLINE | ID: mdl-31691659

RESUMO

Ophthalmologic manifestation of Sjogren's disease (SD) and rheumatoid arthritis (RA) is dry keratoconjunctivitis (dry eye disease; DED). PURPOSE: To study the relationship of polymorphic markers rs7947461 (C/T), rs915956 (C/T), rs4144331 (C/A) of the TRIM21 gene with the severity of DED in patients with RA and SD. MATERIAL AND METHODS: The study included 70 patients with RA (n=27) and SD (n=43). The control group consisted of volunteers without a history of RA or SD (n=35). Alleles of the polymorphic marker C660T rs7947461 of the TRIM21 gene were identified using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method; alleles of the polymorphic marker rs915956 (C/T) and rs4144331 (C/A) of the TRIM21 gene were identified by analyzing DNA melting curves. RESULTS: An association was found between the predisposing genotype (TT) of rs7947461 polymorphic marker and the risk of developing severe DED. The AA genotype of rs4144331 polymorphic marker was found only in severe DED (c2=7.74; OR=17.46, CI95%=1.96-318.38, p=0.02). CONCLUSION: An association was established between rs7947461 (rs660) and rs4144331 and the risk of developing severe DED.


Assuntos
Artrite Reumatoide , Ceratoconjuntivite , Ribonucleoproteínas/genética , Síndrome de Sjogren , Alelos , Artrite Reumatoide/genética , Predisposição Genética para Doença , Genótipo , Humanos , Ceratoconjuntivite/genética , Polimorfismo Genético , Síndrome de Sjogren/genética
2.
Infect Immun ; 87(12)2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31548319

RESUMO

Antibodies are essential for immunity against Ehrlichia chaffeensis, and protective mechanisms involve blocking of ehrlichial attachment or complement and Fcγ-receptor-dependent destruction. In this study, we determined that major outer membrane protein 1 (OMP-19) hypervariable region 1 (HVR1)-specific human monoclonal antibodies (huMAbs) are protective through conventional extracellular neutralization and, more significantly, through a novel intracellular TRIM21-mediated mechanism. Addition of OMP-1-specific huMAb EHRL-15 (IgG1) prevented infection by blocking attachment/entry, a mechanism previously reported; conversely, OMP-1-specific huMAb EHRL-4 (IgG3) engaged intracellular TRIM21 and initiated an immediate innate immune response and rapid intracellular degradation of ehrlichiae. EHRL-4-TRIM21-mediated inhibition was significantly impaired in TRIM21 knockout THP-1 cells. EHRL-4 interacted with cytosolic Fc receptor TRIM21, observed by confocal microscopy and confirmed by co-immunoprecipitation. E. chaffeensis-EHRL-4-TRIM21 complexes caused significant upregulation of proinflammatory cytokine/chemokine transcripts and resulted in rapid (<30 min) nuclear accumulation of NF-κB and TRIM21 and ehrlichial destruction. We investigated the role of TRIM21 in the autophagic clearance of ehrlichiae in the presence of EHRL-4. Colocalization between EHRL-4-opsonized ehrlichiae, polyubiquitinated TRIM21, autophagy regulators (ULK1 and beclin 1) and effectors (LC3 and p62), and lysosome-associated membrane protein 2 (LAMP2) was observed. Moreover, autophagic flux defined by conversion of LC3I to LC3II and accumulation and degradation of p62 was detected, and EHRL-4-mediated degradation of E. chaffeensis was abrogated by the autophagy inhibitor 3-methyladenine. Our results demonstrate that huMAbs are capable of inhibiting E. chaffeensis infection by distinct effector mechanisms: extracellularly by neutralization and intracellularly by engaging TRIM21, which mediates a rapid innate immune response that mobilizes the core autophagy components, triggering localized selective autophagic degradation of ehrlichiae.


Assuntos
Antígenos de Bactérias/imunologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas da Membrana Bacteriana Externa/imunologia , Ehrlichia chaffeensis/imunologia , Ribonucleoproteínas/genética , Adenina/análogos & derivados , Adenina/farmacologia , Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos de Bactérias/genética , Autofagia/imunologia , Aderência Bacteriana/genética , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Ehrlichia chaffeensis/genética , Técnicas de Inativação de Genes , Humanos , Imunidade Humoral/imunologia , NF-kappa B/genética , Células THP-1
3.
Arch Endocrinol Metab ; 63(4): 438-444, 2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31460623

RESUMO

Pubertal timing in humans is determined by complex interactions including hormonal, metabolic, environmental, ethnic, and genetic factors. Central precocious puberty (CPP) is defined as the premature reactivation of the hypothalamic-pituitary-gonadal axis, starting before the ages of 8 and 9 years in girls and boys, respectively; familial CPP is defined by the occurrence of CPP in two or more family members. Pioneering studies have evidenced the participation of genetic factors in pubertal timing, mainly identifying genetic causes of CPP in sporadic and familial cases. In this context, rare activating mutations were identified in genes of the kisspeptin excitatory pathway (KISS1R and KISS1 mutations). More recently, loss-of-function mutations in two imprinted genes (MKRN3 and DLK1) have been identified as important causes of familial CPP, describing novel players in the modulation of the hypothalamic-pituitary-gonadal axis in physiological and pathological conditions. MKRN3 mutations are the most common cause of familial CPP, and patients with MKRN3 mutations present clinical features indistinguishable from idiopathic CPP. Meanwhile, adult patients with DLK1 mutations present high frequency of metabolic alterations (overweight/obesity, early onset type 2 diabetes and hyperlipidemia), indicating that DLK1 may be a novel link between reproduction and metabolism. Arch Endocrinol Metab. 2019;63(4):438-44.


Assuntos
Puberdade Precoce/genética , Proteínas de Ligação ao Cálcio , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Kisspeptinas/genética , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metilação , Mutação , Fenótipo , Puberdade Precoce/etiologia , Receptores de Kisspeptina-1/genética , Ribonucleoproteínas/genética
4.
Exp Appl Acarol ; 78(4): 505-520, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31375950

RESUMO

Phytoseiulus persimilis is one of the most important biological control agents of spider mites. Multiple studies have been conducted on factors affecting its reproduction, but limited research on related molecular mechanisms has been carried out. In this study, RNA interference of three genes, ribosomal protein L11 (RpL11), ribosomal protein S2 (RpS2), and transformer-2 (tra-2), to newly emerged females were performed through oral delivery of double-stranded RNA, and knockdown of target genes was verified using qRT-PCR analysis. When RpL11 or RpS2 was interfered, 42 and 30% P. persimilis individuals either laid no egg or had no egg hatched, whereas the remaining females had their oviposition duration reduced by 31.8 and 49.9%, fecundity reduced by 48.1 and 67.8%, and egg hatching rate reduced by 20.4 and 22.4%, respectively. In addition, offspring sex ratios were significantly male biased especially at low fecundities. When tra-2 was interfered, no significant difference in fecundity was detected, but egg hatching rate reduced by 30.6%. This study verified the possibility of RNA interference in Phytoseiidae through oral delivery, and indicated that RpL11 and RpS2 are involved in egg formation, whereas tra-2 is involved in embryo development in P. persimilis. Phytoseiid mites have different sex determination pathways compared to insects. The present study provides data and evidence at molecular biological level for future research on reproduction and sex determination of phytoseiid mites.


Assuntos
Proteínas de Artrópodes/genética , Ácaros/fisiologia , Interferência de RNA , Animais , Proteínas de Artrópodes/metabolismo , Feminino , Técnicas de Silenciamento de Genes , Ácaros/genética , Reprodução/genética , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/metabolismo
5.
EMBO J ; 38(21): e101365, 2019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31468569

RESUMO

Inflammasomes are potent innate immune signalling complexes that couple cytokine release with pro-inflammatory cell death. However, pathogens have evolved strategies to evade this cell autonomous system. Here, we show how antibodies combine with innate sensors in primary human macrophages to detect viral infection and activate the inflammasome. Our data demonstrate that antibody opsonisation of virions can activate macrophages in multiple ways. In the first, antibody binding of adenovirus causes lysosomal damage, activating NLRP3 to drive inflammasome formation and IL-1ß release. Importantly, this mechanism enhances virion capture but not infection and is accompanied by cell death, denying the opportunity for viral replication. Unexpectedly, we also find that antibody-coated viruses, which successfully escape into the cytosol, trigger a second system of inflammasome activation. These viruses are intercepted by the cytosolic antibody receptor TRIM21 and the DNA sensor cGAS. Together, these sensors stimulate both NLRP3 inflammasome formation and NFκB activation, driving dose-dependent IL-1ß and TNF secretion, without inducing cell death. Our data highlight the importance of cooperativity between multiple sensing networks to expose viruses to the inflammasome pathway, which is particularly important for how our innate immune system responds to infection in the presence of pre-existing immunity.


Assuntos
Infecções por Adenoviridae/imunologia , Anticorpos Antivirais/imunologia , Inflamassomos/imunologia , Macrófagos/imunologia , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Nucleotidiltransferases/metabolismo , Ribonucleoproteínas/metabolismo , Replicação Viral/imunologia , Adenoviridae/genética , Adenoviridae/imunologia , Infecções por Adenoviridae/metabolismo , Infecções por Adenoviridae/virologia , Animais , Células Cultivadas , Humanos , Inflamassomos/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/metabolismo , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Nucleotidiltransferases/genética , Ribonucleoproteínas/genética , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
6.
Nucleic Acids Res ; 47(17): e99, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31299082

RESUMO

Transient expression of the CRISPR/Cas9 machinery will not only reduce risks of mutagenesis from off-target activities, but also decrease possible immune response to Cas9 protein. Building on our recent developing of a system able to package up to 100 copies of Cas9 mRNA in each lentivirus-like particle (LVLP) via the specific interaction between aptamer and aptamer-binding proteins (ABP), here we develop a lentiviral capsid-based bionanoparticle system, which allows efficient packaging of Cas9/sgRNA ribonucleoprotein (RNP). We show that replacing the Tetraloop of sgRNA scaffold with a com aptamer preserves the functions of the guide RNA, and the com-modified sgRNA can package Cas9/sgRNA RNP into lentivirus-like particles via the specific interactions between ABP and aptamer, and sgRNA and Cas9 protein. These RNP bionanoparticles generated Indels on different targets in different cells with efficiencies similar to or better than our recently described Cas9 mRNA LVLPs. The new system showed fast action and reduced off-target rates, and makes it more convenient and efficient in delivering Cas9 RNPs for transient Cas9 expression and efficient genome editing.


Assuntos
Proteína 9 Associada à CRISPR/genética , Sistemas CRISPR-Cas/genética , Edição de Genes/métodos , Técnicas de Transferência de Genes , Ribonucleoproteínas/genética , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Capsídeo/metabolismo , Proteínas do Capsídeo/genética , Vetores Genéticos/genética , Células HEK293 , Humanos , Lentivirus/genética , Nanopartículas/química , Nanopartículas/metabolismo
7.
Int J Mol Sci ; 20(13)2019 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-31323950

RESUMO

Short tandem repeat (STR) or microsatellite, expansions underlie more than 50 hereditary neurological, neuromuscular and other diseases, including myotonic dystrophy types 1 (DM1) and 2 (DM2). Current disease models for DM1 and DM2 propose a common pathomechanism, whereby the transcription of mutant DMPK (DM1) and CNBP (DM2) genes results in the synthesis of CUG and CCUG repeat expansion (CUGexp, CCUGexp) RNAs, respectively. These CUGexp and CCUGexp RNAs are toxic since they promote the assembly of ribonucleoprotein (RNP) complexes or RNA foci, leading to sequestration of Muscleblind-like (MBNL) proteins in the nucleus and global dysregulation of the processing, localization and stability of MBNL target RNAs. STR expansion RNAs also form phase-separated gel-like droplets both in vitro and in transiently transfected cells, implicating RNA-RNA multivalent interactions as drivers of RNA foci formation. Importantly, the nucleation and growth of these nuclear foci and transcript misprocessing are reversible processes and thus amenable to therapeutic intervention. In this review, we provide an overview of potential DM1 and DM2 pathomechanisms, followed by a discussion of MBNL functions in RNA processing and how multivalent interactions between expanded STR RNAs and RNA-binding proteins (RBPs) promote RNA foci assembly.


Assuntos
Processamento Alternativo/genética , Repetições de Microssatélites/genética , Distrofia Miotônica/genética , RNA/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Núcleo Celular/genética , Núcleo Celular/metabolismo , Humanos , Músculo Esquelético/metabolismo , Ribonucleoproteínas/genética , Ribonucleoproteínas/metabolismo
8.
Transgenic Res ; 28(Suppl 2): 61-64, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31321685

RESUMO

Processes of traditional trait development in plants depend on genetic variations derived from spontaneous mutation or artificial random mutagenesis. Limited availability of desired traits in crossable relatives or failure to generate the wanted phenotypes by random mutagenesis led to develop innovative breeding methods that are truly cross-species and precise. To this end, we devised novel methods of precise genome engineering that are characterized to use pre-assembled CRISPR/Cas9 ribonucleoprotein (RNP) complex instead of using nucleic ands or Agrobacterium. We found that our methods successfully engineered plant genomes without leaving any foreign DNA footprint in the genomes. To facilitate introduction of RNP into plant nucleus, we first obtained protoplasts after removing the transfection barrier, cell wall. Whole plants were regenerated from the single cell of protoplasts that has been engineered with the RNP. Pending the improved way of protoplast regeneration technology especially in crop plants, our methods should help develop novel traits in crop plants in relatively short time with safe and precise way.


Assuntos
Sistemas CRISPR-Cas/genética , DNA/genética , Edição de Genes/tendências , Ribonucleoproteínas/genética , Agrobacterium/genética , Genoma de Planta/genética , Mutação , Protoplastos/metabolismo
9.
Pediatr Hematol Oncol ; 36(4): 236-243, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31361176

RESUMO

Here we report a case of refractory macrocytic anemia with a spliceosomal point mutation involving the ZRSR2 gene in a child with Down syndrome (DS). Such mutations have been shown to cause refractory macrocytic anemia and myelodysplastic syndrome (MDS) in elderly individuals. We report the hematological indices of a child with DS and a ZRSR2 spliceosomal mutation. DS is known to produce macrocytic anemia but does not lead to transfusion dependence. In this case, the ZRSR2 mutation was the likely implicating factor for severe transfusion-dependent anemia in a child with DS. The clinical implication of a ZRSR2 mutation in a child with DS has not been previously described and warrants close surveillance to detect potential insidious transformation to MDS.


Assuntos
Anemia Macrocítica/genética , Síndrome de Down/genética , Mutação Puntual , Ribonucleoproteínas/genética , Anemia Macrocítica/sangue , Anemia Macrocítica/terapia , Criança , Síndrome de Down/sangue , Síndrome de Down/terapia , Humanos , Masculino , Ribonucleoproteínas/metabolismo
10.
Genes Dev ; 33(13-14): 741-746, 2019 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-31171702

RESUMO

Site-specific 2'-O-ribose methylation of mammalian rRNAs and RNA polymerase II-synthesized spliceosomal small nuclear RNAs (snRNAs) is mediated by small nucleolar and small Cajal body (CB)-specific box C/D ribonucleoprotein particles (RNPs) in the nucleolus and the nucleoplasmic CBs, respectively. Here, we demonstrate that 2'-O-methylation of the C34 wobble cytidine of human elongator tRNAMet(CAT) is achieved by collaboration of a nucleolar and a CB-specific box C/D RNP carrying the SNORD97 and SCARNA97 box C/D 2'-O-methylation guide RNAs. Methylation of C34 prevents site-specific cleavage of tRNAMet(CAT) by the stress-induced endoribonuclease angiogenin, implicating box C/D guide RNPs in controlling stress-responsive production of putative regulatory tRNA fragments.


Assuntos
Nucléolo Celular/metabolismo , Corpos Enovelados/metabolismo , Citidina/metabolismo , RNA de Transferência/metabolismo , Ribonucleoproteínas/metabolismo , Linhagem Celular , Regulação da Expressão Gênica , Células HeLa , Humanos , Metilação , RNA Nucleolar Pequeno/genética , RNA Nucleolar Pequeno/metabolismo , RNA de Transferência/genética , Ribonuclease Pancreático/metabolismo , Ribonucleoproteínas/genética , Estresse Fisiológico
11.
Mol Cell ; 75(1): 66-75.e5, 2019 07 11.
Artigo em Inglês | MEDLINE | ID: mdl-31175012

RESUMO

Liquid granules rich in intrinsically disordered proteins and RNA play key roles in critical cellular functions such as RNA processing and translation. Many details of the mechanism via which this occurs remain to be elucidated. Motivated by the lacuna in the field and by the prospects of developing de novo artificial granules that provide extrinsic control of translation, we report a bottom-up approach to engineer ribonucleoprotein granules composed of a recombinant RNA-binding IDP that exhibits phase behavior in water. We developed a kinetic model to illustrate that these granules inhibit translation through reversible or irreversible sequestration of mRNA. Within monodisperse droplets capable of transcription and translation, we experimentally demonstrate temporal inhibition of translation by using designer IDPs that exhibit tunable phase behavior. This work lays the foundation for developing artificial granules that promise to further our mechanistic understanding of their naturally occurring counterparts.


Assuntos
Células Artificiais/metabolismo , Grânulos Citoplasmáticos/genética , Proteínas Intrinsicamente Desordenadas/genética , Peptidomiméticos/metabolismo , RNA Mensageiro/genética , Ribonucleoproteínas/genética , Sequência de Aminoácidos , Células Artificiais/citologia , Grânulos Citoplasmáticos/química , Grânulos Citoplasmáticos/metabolismo , Elastina/química , Elastina/genética , Elastina/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Modelos Biológicos , Peptidomiméticos/química , Transição de Fase , Plasmídeos/genética , Plasmídeos/metabolismo , Biossíntese de Proteínas , Engenharia de Proteínas/métodos , RNA/genética , RNA/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Ribonucleoproteínas/química , Ribonucleoproteínas/metabolismo
12.
Mol Med Rep ; 20(2): 951-958, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31173237

RESUMO

Prostate cancer (PCa) is the most common malignancy among males worldwide, and is one of the leading causes of cancer­related mortality. MicroRNAs (miRs) are a type of endogenous, noncoding RNA that serve a key role in pathological processes, and have been demonstrated to be involved in the formation and progression of PCa. Previous studies have reported that miR­106b acts as an oncogene; however, the specific effects of miR­106b on PCa have not been fully elucidated. The present study aimed to investigate the role and underlying molecular mechanisms of miR­106b in the initiation and progression of PCa. In this study, miR­106b was reported to be overexpressed and la­related protein 4B (LARP4B) was downregulated in PCa tissues compared with paracancerous tissues. In addition, LARP4B was identified as a target gene of miR­106b by bioinformatics prediction analysis and a dual luciferase reporter gene assay. Furthermore, MTT, wound healing and Transwell assays were performed to evaluate PCa cell viability, and migration and invasive abilities. The data revealed that inhibition of miR­106b significantly suppressed the viability, migration and invasion of PCa cells. In addition, inhibition of miR­106b significantly suppressed the mRNA and protein expression of cancer­related genes, including matrix metalloproteinase­2, cluster of differentiation 44 and Ki­67, and increased that of the tumor suppressor, mothers against decapentaplegic homolog 2. Collectively, the findings of the present study indicated that miR­106b may target LAR4B to inhibit cancer cell viability, migration and invasion, and may be considered as a novel therapeutic target in PCa.


Assuntos
Autoantígenos/genética , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Próstata/metabolismo , Neoplasias da Próstata/genética , Ribonucleoproteínas/genética , Idoso , Antagomirs/genética , Antagomirs/metabolismo , Autoantígenos/metabolismo , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , Humanos , Receptores de Hialuronatos/genética , Receptores de Hialuronatos/metabolismo , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 2 da Matriz/metabolismo , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Metástase Neoplásica , Estadiamento de Neoplasias , Próstata/patologia , Neoplasias da Próstata/metabolismo , Neoplasias da Próstata/patologia , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Proteína Smad2/genética , Proteína Smad2/metabolismo
13.
Nat Commun ; 10(1): 2806, 2019 06 26.
Artigo em Inglês | MEDLINE | ID: mdl-31243272

RESUMO

CRISPR-Cas adaptive immune systems function to protect bacteria from invasion by foreign genetic elements. The CRISPR-Cas9 system has been widely adopted as a powerful genome-editing tool, and phage-encoded inhibitors, known as anti-CRISPRs, offer a means of regulating its activity. Here, we report the crystal structures of anti-CRISPR protein AcrIIC2Nme alone and in complex with Nme1Cas9. We demonstrate that AcrIIC2Nme inhibits Cas9 through interactions with the positively charged bridge helix, thereby preventing sgRNA loading. In vivo phage plaque assays and in vitro DNA cleavage assays show that AcrIIC2Nme mediates its activity through a large electronegative surface. This work shows that anti-CRISPR activity can be mediated through the inhibition of Cas9 complex assembly.


Assuntos
Bacteriófagos/metabolismo , Proteína 9 Associada à CRISPR/antagonistas & inibidores , Sistemas CRISPR-Cas , Ribonucleoproteínas/metabolismo , Proteínas Virais/farmacologia , Escherichia coli/metabolismo , Edição de Genes , Regulação Bacteriana da Expressão Gênica , Neisseria/virologia , Ribonucleoproteínas/genética , Proteínas Virais/metabolismo
14.
Eur J Endocrinol ; 181(2): 103-119, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31200363

RESUMO

Context: Congenital hypogonadotropic hypogonadism (CHH) is a rare condition caused by GnRH deficiency. Several genes have been associated with the pathogenesis of CHH, but most cases still remain without a molecular diagnosis. The advent of next-generation sequencing (NGS) has allowed the simultaneous genotyping of several regions, faster, making possible the extension of the genetic knowledge of CHH. Objective: Genetic characterization of a large cohort of Brazilian CHH patients. Design and patients: A cohort of 130 unrelated patients (91 males, 39 females) with CHH (75 normosmic CHH, 55 Kallmann syndrome) was studied using a panel containing 36 CHH-associated genes. Results: Potential pathogenic or probably pathogenic variants were identified in 43 (33%) CHH patients. The genes ANOS1, FGFR1 and GNRHR were the most frequently affected. A novel homozygous splice site mutation was identified in the GNRH1 gene and a deletion of the entire coding sequence was identified in SOX10. Deleterious variants in the IGSF10 gene were identified in two patients with reversible normosmic CHH. Notably, 6.9% of the patients had rare variants in more than one gene. Rare variants were also identified in SPRY4, IL17RD, FGF17, IGSF1 and FLRT3 genes. Conclusions: This is a large study of the molecular genetics of CHH providing new genetic findings for this complex and heterogeneous genetic condition. NGS has been shown to be a fast, reliable and effective tool in the molecular diagnosis of congenital CHH and being able to targeting clinical genetic testing in the future.


Assuntos
Hipogonadismo/congênito , Hipogonadismo/genética , Mutação , Adulto , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Brasil/epidemiologia , Proteínas de Transporte/genética , Estudos de Coortes , Análise Mutacional de DNA , Feminino , Predisposição Genética para Doença , Testes Genéticos , Glicoproteínas/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hipogonadismo/diagnóstico , Hipogonadismo/epidemiologia , Imunoglobulinas/genética , Síndrome de Kallmann/diagnóstico , Síndrome de Kallmann/epidemiologia , Síndrome de Kallmann/genética , Fator de Transcrição MSX1/genética , Masculino , Proteínas de Membrana/genética , Fatores de Transcrição Otx/genética , Linhagem , Receptores de Grelina/genética , Ribonucleoproteínas/genética , Adulto Jovem
15.
Nat Commun ; 10(1): 2229, 2019 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-31110176

RESUMO

Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive, and highly lethal lung disease with unknown etiology and poor prognosis. IPF patients die within 2 years after diagnosis mostly due to respiratory failure. Current treatments against IPF aim to ameliorate patient symptoms and to delay disease progression. Unfortunately, therapies targeting the causes of or reverting IPF have not yet been developed. Here we show that reduced levels of miRNA lethal 7d (MIRLET7D) in IPF compromise epigenetic gene silencing mediated by the ribonucleoprotein complex MiCEE. In addition, we find that hyperactive EP300 reduces nuclear HDAC activity and interferes with MiCEE function in IPF. Remarkably, EP300 inhibition reduces fibrotic hallmarks of in vitro (patient-derived primary fibroblast), in vivo (bleomycin mouse model), and ex vivo (precision-cut lung slices, PCLS) IPF models. Our work provides the molecular basis for therapies against IPF using EP300 inhibition.


Assuntos
Proteína p300 Associada a E1A/metabolismo , Histona Desacetilase 1/metabolismo , Fibrose Pulmonar Idiopática/patologia , MicroRNAs/metabolismo , Ribonucleoproteínas/metabolismo , Animais , Bleomicina/toxicidade , Núcleo Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Células Cultivadas , Modelos Animais de Doenças , Proteína p300 Associada a E1A/antagonistas & inibidores , Fibroblastos , Inativação Gênica , Histona Desacetilase 2/metabolismo , Humanos , Fibrose Pulmonar Idiopática/induzido quimicamente , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/genética , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Camundongos , Cultura Primária de Células , Ribonucleoproteínas/genética
16.
Folia Histochem Cytobiol ; 57(2): 56-63, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31112282

RESUMO

INTRODUCTION: Long interspersed nuclear elements-1 (L1), as the only one self-active retrotransposon of the mobile element, was found to be generally activated in tumor cells. The 5'UTR of L1 (L1-5'UTR) contains both sense and antisense bidirectional promoters, transcription products of which can generate double-strand RNA (dsRNA). In addition, L1-ORF1p, a dsRNA binding protein encoded by L1, is considered to engage in some RNA-protein (RNP) formation. Ago2, one of the RISC components, can bind to dsRNA to form RISC complex, but its role in L1 regulation still remains unclear. Due that the 5'UTR of L1 (L1-5'UTR) contains both sense and antisense bidirectional promoters, so the activities in both string were identified. A dsRNA-mediated regulation of L1-5'UTR, with the feedback regulation of L1-ORF1p as well as other key molecules engaged (Ago1-4) in this process, was also investigated. MATERIAL AND METHODS: Genomic DNA was extracted from HEK293 cells and subjected to L1-5'UTR prepa-ration by PCR. Report gene system pIRESneo with SV40 promoter was employed. The promoter activities of different regions in L1-5'UTR were identified by constructing these regions into pIRESneo, which SV40 region was removed prior, to generate different recombinant plasmids. The promoter activities in recombinant plasmids were detected by the luciferase expression assay. Western blot and co-immunoprecipitation were employed to identify proteins expression and protein-protein interaction respectively. RESULTS: Ago2 is a member of Agos family, which usually forms a RISC complex with si/miRNA and is involved in post- transcriptional regulation of many genes. Here L1-ORF1p and Ago2 conducts a regulation as a negative feedback for L1-5'UTR sense promoter. L1-ORF1p could form the immune complexes with Ago1, Ago2 and Ago4, respectively. CONCLUSIONS: L1-5'UTR harbors both sense and antisense promoter activity and a dsRNA-mediated regulation is responsible for L1-5'UTR regulation. Agos proteins and L1-ORF1p were engaged in this process.


Assuntos
Regiões 5' não Traduzidas , Proteínas Argonauta/genética , Regulação da Expressão Gênica , Elementos Nucleotídeos Longos e Dispersos , RNA Interferente Pequeno/genética , Ribonucleoproteínas/genética , Sequência de Bases , Células HEK293 , Humanos , Mutação , Regiões Promotoras Genéticas
17.
Medicine (Baltimore) ; 98(21): e15743, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31124956

RESUMO

BACKGROUND: Gene mutations with important prognostic role have been identified in patients with myelodysplastic syndrome (MDS). We performed a meta-analysis to investigate the effects of RNA splicing machinery gene mutations on prognosis of MDS patients. METHODS: We searched English database including PubMed, Embase, Cochrane Library for literatures published within recent 10 years on the effect of RNA splicing machinery genes in MDS. Revman version 5.2 software was used for all the statistical processing. We calculated risk ratio and 95% confidence interval (CI) of continuous variables, and find hazard ratio (HR) and 95% CI of time-to-event data. RESULTS: We included 19 studies enrolling 4320 patients. There is a significant superior overall survival (OS) in splicing factor 3b, subunit 1 (SF3B1)-mutation group compared to unmutated group (HR = 0.58, 95% CI: 0.5-0.67, P < .00001); OS decreased significantly in serine/arginine-rich splicing factor 2/ U2 auxiliary factor protein 1 (SRSF2/U2AF1) mutation group compared to unmutated group, (HR = 1.62, 95% CI: 1.34-1.97, P < .00001 and HR = 1.61, 95% CI: 1.35-1.9, P < .00001, respectively). In terms of leukemia-free survival (LFS), the group with SF3B1 mutation had better outcome than unmutated group, HR = 0.63 (95% CI: 0.53-0.75, P < .00001). Other RNA splicing gene mutation group showed significant poor LFS than unmutated groups, (HR = 1.89, 95% CI: 1.6-2.23, P < .00001; HR = 2.77, 95% CI: 2.24-3.44, P < .00001; HR = 1.48, 95% CI: 1.08-2.03, P < .00001; for SRSF2, U2AF1, and zinc finger CCCH-type, RNA binding motif and serine/arginine rich 2 [ZRSR2], respectively). As for subgroup of low- or intermediate-1-IPSS risk MDS, SRSF2, and U2AF1 mutations were related to poor OS. (HR = 1.83, 95% CI: 1.43-2.35, P < .00001; HR = 2.11, 95% CI: 1.59-2.79, P < .00001 for SRSF2 and U2AF1, respectively). SRSF2 and U2AF1 mutations were strongly associated with male patients. SF3B1 mutation was strongly associated with disease staging. CONCLUSION: This meta-analysis indicates a positive effect of SF3B1 and an adverse prognostic effect of SRSF2, U2AF1, and ZRSR2 mutations in patients with MDS. Mutations of RNA splicing genes have important effects on the prognosis of MDS.


Assuntos
Síndromes Mielodisplásicas/genética , Processamento de RNA/genética , Humanos , Proteínas Nucleares/genética , Fosfoproteínas/genética , Prognóstico , Fatores de Processamento de RNA/genética , Ribonucleoproteínas/genética , Fatores de Processamento de Serina-Arginina/genética , Índice de Gravidade de Doença , Fatores Sexuais , Fator de Processamento U2AF/genética , Análise de Sobrevida
18.
Nucleic Acids Res ; 47(14): 7605-7617, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31127285

RESUMO

Group II introns are self-splicing mobile genetic retroelements. The spliced intron RNA and the intron-encoded protein (IEP) form ribonucleoprotein particles (RNPs) that recognize and invade specific DNA target sites. The IEP is a reverse transcriptase/maturase that may bear a C-terminal endonuclease domain enabling the RNP to cleave the target DNA strand to prime reverse transcription. However, some mobile introns, such as RmInt1, lack the En domain but nevertheless retrohome efficiently to transient single-stranded DNA target sites at a DNA replication fork. Their mobility is associated with host DNA replication, and they use the nascent lagging strand as a primer for reverse transcription. We searched for proteins that interact with RmInt1 RNPs and direct these RNPs to the DNA replication fork. Co-immunoprecipitation assays suggested that DnaN (the ß-sliding clamp), a component of DNA polymerase III, interacts with the protein component of the RmInt1 RNP. Pulldown assays, far-western blots and biolayer interferometry supported this interaction. Peptide binding assays also identified a putative DnaN-interacting motif in the RmInt1 IEP structurally conserved in group II intron IEPs. Our results suggest that intron RNP interacts with the ß-sliding clamp of the DNA replication machinery, favouring reverse splicing into the transient ssDNA at DNA replication forks.


Assuntos
Proteínas de Bactérias/genética , Replicação do DNA/genética , DNA Polimerase Dirigida por DNA/genética , Processamento de RNA , Retroelementos/genética , Ribonucleoproteínas/genética , Sinorhizobium meliloti/genética , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Inteínas/genética , Íntrons/genética , Modelos Genéticos , Ligação Proteica , DNA Polimerase Dirigida por RNA/genética , DNA Polimerase Dirigida por RNA/metabolismo , Ribonucleoproteínas/metabolismo , Sinorhizobium meliloti/metabolismo
19.
RNA Biol ; 16(7): 930-939, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30943851

RESUMO

The RmInt1 group II intron is an efficient self-splicing mobile retroelement that catalyzes its own excision as lariat, linear and circular molecules. In vivo, the RmInt1 lariat and the reverse transcriptase (IEP) it encodes form a ribonucleoprotein particle (RNP) that recognizes the DNA target for site-specific full intron insertion via a two-step reverse splicing reaction. RNPs containing linear group II intron RNA are generally thought to be unable to complete the reverse splicing reaction. Here, we show that reconstituted in vitro RNPs containing linear RmInt1 ΔORF RNA can mediate the cleavage of single-stranded DNA substrates in a very precise manner with the attachment of the intron RNA to the 3´exon as the first step of a reverse splicing reaction. Notably, we also observe molecules in which the 5´exon is linked to the RmInt1 RNA, suggesting the completion of the reverse splicing reaction, albeit rather low and inefficiently. That process depends on DNA target recognition and can be successful completed by RmInt1 RNPs with linear RNA displaying 5´ modifications.


Assuntos
Clivagem do DNA , Íntrons/genética , Processamento de RNA/genética , Ribonucleoproteínas/genética , Sequência de Bases , DNA Bacteriano/metabolismo , RNA Bacteriano/genética , Ribonucleoproteínas/metabolismo , Sinorhizobium meliloti/genética , Fatores de Tempo
20.
PLoS One ; 14(4): e0215688, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31009498

RESUMO

Reproduction is a process that is extremely sensitive to changes in nutritional status. The nutritional control of oogenesis via insulin signaling has been reported; however, the mechanism underlying its sensitivity and tissue specificity has not been elucidated. Here, we determined that Drosophila Makorin RING finger protein 1 gene (Mkrn1) functions in the metabolic regulation of oogenesis. Mkrn1 was endogenously expressed at high levels in ovaries and Mkrn1 knockout resulted in female sterility. Mkrn1-null egg chambers were previtellogenic without egg production. FLP-FRT mosaic analysis revealed that Mkrn1 is essential in germline cells, but not follicle cells, for ovarian function. As well, AKT phosphorylation via insulin signaling was greatly reduced in the germline cells, but not the follicle cells, of the mutant clones in the ovaries. Furthermore, protein-rich diet elevated Mkrn1 protein levels, without increased mRNA levels. The p-AKT and p-S6K levels, downstream targets of insulin/Tor signaling, were significantly increased by a nutrient-rich diet in wild-type ovaries whereas those were low in Mkrn1exS compared to wild-type ovaries. Taken together, our results suggest that nutrient availability upregulates the Mkrn1 protein, which acts as a positive regulator of insulin signaling to confer sensitivity and tissue specificity in the ovaries for proper oogenesis based on nutritional status.


Assuntos
Proteínas de Drosophila/metabolismo , Insulina Regular Humana/metabolismo , Insulina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas na Dieta/administração & dosagem , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Feminino , Insulina Regular Humana/genética , Mutação , Proteínas do Tecido Nervoso/genética , Oócitos/citologia , Oócitos/metabolismo , Oogênese/genética , Ovário/citologia , Ovário/efeitos dos fármacos , Ovário/metabolismo , Ribonucleoproteínas/genética
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