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1.
Am J Hum Genet ; 105(3): 640-657, 2019 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-31402090

RESUMO

The identification of genetic variants implicated in human developmental disorders has been revolutionized by second-generation sequencing combined with international pooling of cases. Here, we describe seven individuals who have diverse yet overlapping developmental anomalies, and who all have de novo missense FBXW11 variants identified by whole exome or whole genome sequencing and not reported in the gnomAD database. Their phenotypes include striking neurodevelopmental, digital, jaw, and eye anomalies, and in one individual, features resembling Noonan syndrome, a condition caused by dysregulated RAS signaling. FBXW11 encodes an F-box protein, part of the Skp1-cullin-F-box (SCF) ubiquitin ligase complex, involved in ubiquitination and proteasomal degradation and thus fundamental to many protein regulatory processes. FBXW11 targets include ß-catenin and GLI transcription factors, key mediators of Wnt and Hh signaling, respectively, critical to digital, neurological, and eye development. Structural analyses indicate affected residues cluster at the surface of the loops of the substrate-binding domain of FBXW11, and the variants are predicted to destabilize the protein and/or its interactions. In situ hybridization studies on human and zebrafish embryonic tissues demonstrate FBXW11 is expressed in the developing eye, brain, mandibular processes, and limb buds or pectoral fins. Knockdown of the zebrafish FBXW11 orthologs fbxw11a and fbxw11b resulted in embryos with smaller, misshapen, and underdeveloped eyes and abnormal jaw and pectoral fin development. Our findings support the role of FBXW11 in multiple developmental processes, including those involving the brain, eye, digits, and jaw.

2.
PLoS One ; 9(2): e88217, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-24558381

RESUMO

The purpose of this study was to expand our knowledge of small RNAs, which are known to function within protein complexes to modulate the transcriptional output of the cell. Here we describe two previously unrecognized, small RNAs, termed pY RNA1-s1 and pY RNA1-s2 (processed Y RNA1-stem -1 and -2), thereby expanding the list of known small RNAs. pY RNA1-s1 and pY RNA1-s2 were discovered by RNA sequencing and found to be 20-fold more abundant in the retina than in 14 other rat tissues. Retinal expression of pY RNAs is highly conserved, including expression in the human retina, and occurs in all retinal cell layers. Mass spectrometric analysis of pY RNA1-S2 binding proteins in retina indicates that pY RNA1-s2 selectively binds the nuclear matrix protein Matrin 3 (Matr3) and to a lesser degree to hnrpul1 (heterogeneous nuclear ribonucleoprotein U-like protein). In contrast, pY RNA1-s1 does not bind these proteins. Accordingly, the molecular mechanism of action of pY RNA1-s2 is likely be through an action involving Matr3; this 95 kDa protein has two RNA recognition motifs (RRMs) and is implicated in transcription and RNA-editing. The high affinity binding of pY RNA1-s2 to Matr3 is strongly dependent on the sequence of the RNA and both RRMs of Matr3. Related studies also indicate that elements outside of the RRM region contribute to binding specificity and that phosphorylation enhances pY RNA-s2/Matr3 binding. These observations are of significance because they reveal that a previously unrecognized small RNA, pY RNA1-s2, binds selectively to Matr3. Hypothetically, pY RNA1-S2 might act to modulate cellular function through this molecular mechanism. The retinal enrichment of pY RNA1-s2 provides reason to suspect that the pY RNA1-s2/Matr3 interaction could play a role in vision.


Assuntos
Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Nucleares/metabolismo , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Retina/metabolismo , Adulto , Motivos de Aminoácidos , Animais , Sequência de Bases , Bovinos , Galinhas , Feminino , Ribonucleoproteínas Nucleares Heterogêneas/metabolismo , Humanos , Macaca mulatta , Masculino , Espectrometria de Massas , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Fosforilação , Glândula Pineal/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Interferência de RNA , Ratos Sprague-Dawley , Ovinos , Distribuição Tecidual
3.
Mol Endocrinol ; 27(11): 1840-55, 2013 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-24085820

RESUMO

Hypothalamic GnRH is the primary regulator of reproduction in vertebrates, acting via the G protein-coupled GnRH receptor (GnRHR) in pituitary gonadotrophs to control synthesis and release of gonadotropins. To identify elements of the GnRHR-coupled gene network, GnRH was applied in a pulsatile manner for 6 hours to a mixed population of perifused pituitary cells from cycling females, mRNA was extracted, and RNA sequencing analysis was performed. This revealed 83 candidate-regulated genes, including a large number coding for secreted proteins. Most notably, GnRH induces a greater than 600-fold increase in expression of dentin matrix protein-1 (Dmp1), one of five members of the small integrin-binding ligand N-linked glycoprotein gene family. The Dmp1 response is mediated by the GnRHR, not elicited by other hypothalamic releasing factors, and is approximately 20-fold smaller in adult male pituitary cells. The sex-dependent Dmp1 response is established during the peripubertal period and independent of the developmental pattern of Gnrhr expression. In vitro, GnRH-induced expression of this gene is coupled with release of DMP1 in extracellular medium through the regulated secretory pathway. In vivo, pituitary Dmp1 expression in identified gonadotrophs is elevated after ovulation. Cell signaling studies revealed that the GnRH induction of Dmp1 is mediated by the protein kinase C signaling pathway and reflects opposing roles of ERK1/2 and p38 MAPK; in addition, the response is facilitated by progesterone. These results establish that DMP1 is a novel secretory protein of female rat gonadotrophs, the synthesis and release of which are controlled by the hypothalamus through the GnRHR signaling pathway. This advance raises intriguing questions about the intrapituitary and downstream effects of this new player in GnRH signaling.


Assuntos
Proteínas da Matriz Extracelular/metabolismo , Gonadotrofos/metabolismo , Hormônio Liberador de Gonadotropina/fisiologia , Fosfoproteínas/metabolismo , Ativação Transcricional , Animais , Células COS , Células Cultivadas , Quimiocinas/genética , Quimiocinas/metabolismo , Estro/metabolismo , Proteínas da Matriz Extracelular/genética , Feminino , Sistema de Sinalização das MAP Quinases , Masculino , Fosfoproteínas/genética , Hipófise/citologia , Hipófise/metabolismo , Proteína Quinase C/metabolismo , Ratos , Ratos Sprague-Dawley , Caracteres Sexuais , Transcriptoma
4.
Proc Natl Acad Sci U S A ; 109(33): 13319-24, 2012 Aug 14.
Artigo em Inglês | MEDLINE | ID: mdl-22864914

RESUMO

Long noncoding RNAs (lncRNAs) play a broad range of biological roles, including regulation of expression of genes and chromosomes. Here, we present evidence that lncRNAs are involved in vertebrate circadian biology. Differential night/day expression of 112 lncRNAs (0.3 to >50 kb) occurs in the rat pineal gland, which is the source of melatonin, the hormone of the night. Approximately one-half of these changes reflect nocturnal increases. Studies of eight lncRNAs with 2- to >100-fold daily rhythms indicate that, in most cases, the change results from neural stimulation from the central circadian oscillator in the suprachiasmatic nucleus (doubling time = 0.5-1.3 h). Light exposure at night rapidly reverses (halving time = 9-32 min) levels of some of these lncRNAs. Organ culture studies indicate that expression of these lncRNAs is regulated by norepinephrine acting through cAMP. These findings point to a dynamic role of lncRNAs in the circadian system.


Assuntos
Ritmo Circadiano/genética , Glândula Pineal/metabolismo , RNA não Traduzido/genética , Animais , Bucladesina/farmacologia , Ritmo Circadiano/efeitos dos fármacos , Biologia Computacional , Regulação da Expressão Gênica/efeitos dos fármacos , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/metabolismo , Norepinefrina/farmacologia , Glândula Pineal/efeitos dos fármacos , RNA não Traduzido/metabolismo , Ratos , Ratos Sprague-Dawley , Sinapses/efeitos dos fármacos , Sinapses/metabolismo
5.
J Biol Chem ; 287(30): 25312-24, 2012 Jul 20.
Artigo em Inglês | MEDLINE | ID: mdl-22908386

RESUMO

MicroRNAs (miRNAs) play a broad range of roles in biological regulation. In this study, rat pineal miRNAs were profiled for the first time, and their importance was evaluated by focusing on the main function of the pineal gland, melatonin synthesis. Massively parallel sequencing and related methods revealed the miRNA population is dominated by a small group of miRNAs as follows: ~75% is accounted for by 15 miRNAs; miR-182 represents 28%. In addition to miR-182, miR-183 and miR-96 are also highly enriched in the pineal gland, a distinctive pattern also found in the retina. This effort also identified previously unrecognized miRNAs and other small noncoding RNAs. Pineal miRNAs do not exhibit a marked night/day difference in abundance with few exceptions (e.g. 2-fold night/day differences in the abundance of miR-96 and miR-182); this contrasts sharply with the dynamic 24-h pattern that characterizes the pineal transcriptome. During development, the abundance of most pineal gland-enriched miRNAs increases; however, there is a marked decrease in at least one, miR-483. miR-483 is a likely regulator of melatonin synthesis, based on the following. It inhibits melatonin synthesis by pinealocytes in culture; it acts via predicted binding sites in the 3"-UTR of arylalkylamine N-acetyltransferase (Aanat) mRNA, the penultimate enzyme in melatonin synthesis, and it exhibits a developmental profile opposite to that of Aanat transcripts. Additionally, a miR-483 targeted antagonist increased melatonin synthesis in neonatal pinealocytes. These observations support the hypothesis that miR-483 suppresses Aanat mRNA levels during development and that the developmental decrease in miR-483 abundance promotes melatonin synthesis.


Assuntos
Regiões 3' não Traduzidas/fisiologia , Arilalquilamina N-Acetiltransferase/biossíntese , Melatonina/biossíntese , MicroRNAs/metabolismo , Glândula Pineal/metabolismo , Animais , Células Cultivadas , Células HEK293 , Humanos , Especificidade de Órgãos/fisiologia , Glândula Pineal/citologia , Glândula Pineal/crescimento & desenvolvimento , Ratos , Ratos Sprague-Dawley
6.
FEMS Microbiol Ecol ; 61(1): 65-73, 2007 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-17466026

RESUMO

Acaryochloris marina strains have been isolated from several varied locations and habitats worldwide demonstrating a diverse and dynamic ecology. In this study, the whole cell photophysiologies of strain MBIC11017, originally isolated from a colonial ascidian, and the free-living epilithic strain CCMEE5410 are analyzed by absorbance and fluorescence spectroscopy, laser scanning confocal microscopy, sodium dodecyl sulfate polyacrylamide gel electrophoresis and subsequent protein analysis. We demonstrate pigment adaptation in MBIC11017 and CCMEE5410 under different light regimes. We show that the higher the incident growth light intensity for both strains, the greater the decrease in their chlorophyll d content. However, the strain MBIC11017 loses its phycobiliproteins relative to its chlorophyll d content when grown at light intensities of 40 microE m(-2) s(-1) without shaking and 100 microE m(-2) s(-1) with shaking. We also conclude that phycobiliproteins are absent in the free-living strain CCMEE5410.


Assuntos
Adaptação Fisiológica , Clorofila/metabolismo , Cianobactérias/efeitos da radiação , Fotossíntese/efeitos da radiação , Ficobiliproteínas/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/metabolismo , Clorofila/efeitos da radiação , Cianobactérias/metabolismo , Cianobactérias/fisiologia , Ecossistema , Microscopia Confocal , Dados de Sequência Molecular , Fotossíntese/fisiologia , Ficobiliproteínas/efeitos da radiação , Espectrometria de Fluorescência , Espectrofotometria , Simbiose/fisiologia
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