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1.
Bioorg Med Chem ; 99: 117596, 2024 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-38232459

RESUMO

Codification of DNA Encoded Libraries (DELs) is critical for successful ligand identification of molecules that bind a protein of interest (POI). There are different encoding strategies that permit, for instance, the customization of a DEL for testing single or dual pharmacophores (single strand DNA) or for producing and screening large diversity libraries of small molecules (double strand DNA). Both approaches challenges, either from the synthetic and encoding point of view, or from the selection methodology to be utilized for the screening. The Head-Piece contains the DNA sequence that is attached to a chemical compound, allowing the encoding of each molecule with a unique DNA tag. Designing the Head-Piece for a DNA-encoded library involves careful consideration of several key aspects including DNA barcode identity, sequence length and attachment chemistry. Here we describe a double stranded DNA versatile Head-Piece that can be used for the generation of single or dual pharmacophore libraries, but also shows other advanced DEL functionalities, stability and enlarged encoding capacity.


Assuntos
Descoberta de Drogas , Bibliotecas de Moléculas Pequenas , Descoberta de Drogas/métodos , Bibliotecas de Moléculas Pequenas/química , DNA/química , Biblioteca Gênica , DNA de Cadeia Simples
2.
J Exp Bot ; 74(7): 2405-2415, 2023 04 09.
Artigo em Inglês | MEDLINE | ID: mdl-36579724

RESUMO

Subcellular mRNA quantities and spatial distributions are fundamental for driving gene regulatory programmes. Single molecule RNA fluorescence in situ hybridization (smFISH) uses fluorescent probes to label individual mRNA molecules, thereby facilitating both localization and quantitative studies. Validated reference mRNAs function as positive controls and are required for calibration. Here we present selection criteria for the first set of Arabidopsis smFISH reference genes. Following sequence and transcript data assessments, four mRNA probe sets were selected for imaging. Transcript counts per cell, correlations with cell size, and corrected fluorescence intensities were all calculated for comparison. In addition to validating reference probe sets, we present sample preparation steps that can retain green fluorescent protein fluorescence, thereby providing a method for simultaneous RNA and protein detection. In summary, our reference gene analyses, modified protocol, and simplified quantification method together provide a firm foundation for future quantitative single molecule RNA studies in Arabidopsis root apical meristem cells.


Assuntos
Arabidopsis , RNA , RNA/genética , Arabidopsis/genética , Hibridização in Situ Fluorescente/métodos , RNA Mensageiro/genética , Regulação da Expressão Gênica
3.
RNA Biol ; 20(1): 836-846, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-37953645

RESUMO

The long noncoding RNA (lncR) ANRIL in the human genome is an established genetic risk factor for atherosclerosis, periodontitis, diabetes, and cancer. However, the regulatory role of lncR-ANRIL in bone and adipose tissue metabolism remains unclear. To elucidate the function of lncRNA ANRIL in a mouse model, we investigated its ortholog, AK148321 (referred to as lncR-APDC), located on chr4 of the mouse genome, which is hypothesized to have similar biological functions to ANRIL. We initially revealed that lncR-APDC in mouse bone marrow cells (BMSCs) and lncR-ANRIL in human osteoblasts (hFOBs) are both increased during early osteogenesis. Subsequently, we examined the osteogenesis, adipogenesis, osteoclastogenesis function with lncR-APDC deletion/overexpression cell models. In vivo, we compared the phenotypic differences in bone and adipose tissue between APDC-KO and wild-type mice. Our findings demonstrated that lncR-APDC deficiency impaired osteogenesis while promoting adipogenesis and osteoclastogenesis. Conversely, the overexpression of lncR-APDC stimulated osteogenesis, but impaired adipogenesis and osteoclastogenesis. Furthermore, KDM6B was downregulated with lncR-APDC deficiency and upregulated with overexpression. Through binding-site analysis, we identified miR-99a as a potential target of lncR-APDC. The results suggest that lncR-APDC exerts its osteogenic function via miR-99a/KDM6B/Hox pathways. Additionally, osteoclasto-osteogenic imbalance was mediated by lncR-APDC through MAPK/p38 and TLR4/MyD88 activation. These findings highlight the pivotal role of lncR-APDC as a key regulator in bone and fat tissue metabolism. It shows potential therapeutic for addressing imbalances in osteogenesis, adipogenesis, and osteoclastogenesis.


Assuntos
MicroRNAs , RNA Longo não Codificante , Humanos , Camundongos , Animais , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Osso e Ossos/metabolismo , Osteogênese/genética , Tecido Adiposo/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , Histona Desmetilases com o Domínio Jumonji
4.
Nucleic Acids Res ; 48(4): 1800-1810, 2020 02 28.
Artigo em Inglês | MEDLINE | ID: mdl-31930333

RESUMO

Steroid hormones are pivotal modulators of pathophysiological processes in many organs, where they interact with nuclear receptors to regulate gene transcription. However, our understanding of hormone action at the single cell level remains incomplete. Here, we focused on estrogen stimulation of the well-characterized GREB1 and MYC target genes that revealed large differences in cell-by-cell responses, and, more interestingly, between alleles within the same cell, both over time and hormone concentration. We specifically analyzed the role of receptor level and activity state during allele-by-allele regulation and found that neither receptor level nor activation status are the determinant of maximal hormonal response, indicating that additional pathways are potentially in place to modulate cell- and allele-specific responses. Interestingly, we found that a small molecule inhibitor of the arginine methyltransferases CARM1 and PRMT6 was able to increase, in a gene specific manner, the number of active alleles/cell before and after hormonal stimulation, suggesting that mechanisms do indeed exist to modulate hormone receptor responses at the single cell and allele level.


Assuntos
Proteínas de Neoplasias/genética , Proteínas Nucleares/genética , Proteína-Arginina N-Metiltransferases/genética , Proteínas Proto-Oncogênicas c-myc/genética , Transcrição Gênica , Estrogênios/metabolismo , Hormônios Esteroides Gonadais/genética , Histona Acetiltransferases/genética , Humanos , Conformação Molecular , Proteínas Nucleares/antagonistas & inibidores , Ligação Proteica/genética , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Análise de Célula Única
5.
Proc Natl Acad Sci U S A ; 114(8): E1509-E1518, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28193854

RESUMO

Spinal muscular atrophy (SMA) is a neurodegenerative disease characterized by progressive motor neuron loss and caused by mutations in SMN1 (Survival Motor Neuron 1). The disease severity inversely correlates with the copy number of SMN2, a duplicated gene that is nearly identical to SMN1. We have delineated a mechanism of transcriptional regulation in the SMN2 locus. A previously uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 (SMN-AS1), represses SMN2 expression by recruiting the Polycomb Repressive Complex 2 (PRC2) to its locus. Chemically modified oligonucleotides that disrupt the interaction between SMN-AS1 and PRC2 inhibit the recruitment of PRC2 and increase SMN2 expression in primary neuronal cultures. Our approach comprises a gene-up-regulation technology that leverages interactions between lncRNA and PRC2. Our data provide proof-of-concept that this technology can be used to treat disease caused by epigenetic silencing of specific loci.


Assuntos
Atrofia Muscular Espinal/terapia , Oligonucleotídeos/genética , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/metabolismo , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Animais , Linhagem Celular , Modelos Animais de Doenças , Éxons/genética , Fibroblastos , Dosagem de Genes , Terapia Genética/métodos , Humanos , Camundongos , Terapia de Alvo Molecular/métodos , Neurônios Motores/metabolismo , Atrofia Muscular Espinal/genética , Mutação Puntual , Complexo Repressor Polycomb 2/genética , RNA Longo não Codificante/genética , Proteína 1 de Sobrevivência do Neurônio Motor/genética , Ativação Transcricional/genética , Regulação para Cima
6.
Proc Natl Acad Sci U S A ; 110(6): 2169-74, 2013 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-23345430

RESUMO

The polyamines, putrescine, spermidine, and spermine, are essential polycations, intimately involved in the regulation of cellular proliferation. Although polyamines exert dynamic effects on the conformation of nucleic acids and macromolecular synthesis in vitro, their specific functions in vivo are poorly understood. We investigated the cellular function of polyamines by overexpression of a key catabolic enzyme, spermidine/spermine N(1)-acetyltransferase 1 (SAT1) in mammalian cells. Transient cotransfection of HeLa cells with GFP and SAT1 vectors suppressed GFP protein expression without lowering its mRNA level, an indication that the block in GFP expression was not at transcription, but at translation. Fluorescence single-cell imaging also revealed specific inhibition of endogenous protein synthesis in the SAT1 overexpressing cells, without any inhibition of synthesis of DNA or RNA. Overexpression of SAT1 using a SAT1 adenovirus led to rapid depletion of cellular spermidine and spermine, total inhibition of protein synthesis, and growth arrest within 24 h. The SAT1 effect is most likely due to depletion of spermidine and spermine, because stable polyamine analogs that are not substrates for SAT1 restored GFP and endogenous protein synthesis. Loss of polysomes with increased 80S monosomes in the polyamine-depleted cells suggests a direct role for polyamines in translation initiation. Our data provide strong evidence for a primary function of polyamines, spermidine and spermine, in translation in mammalian cells.


Assuntos
Proliferação de Células , Biossíntese de Proteínas , Espermidina/metabolismo , Espermina/metabolismo , Acetiltransferases/genética , Acetiltransferases/metabolismo , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Polirribossomos/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
7.
Nucleic Acids Res ; 41(7): 4036-48, 2013 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-23444138

RESUMO

Nuclear receptors (NRs) are central regulators of pathophysiological processes; however, how their responses intertwine is still not fully understood. The aim of this study was to determine whether and how steroid NRs can influence each other's activity under co-agonist treatment. We used a unique system consisting of a multicopy integration of an estrogen receptor responsive unit that allows direct visualization and quantification of estrogen receptor alpha (ERα) DNA binding, co-regulator recruitment and transcriptional readout. We find that ERα DNA loading is required for other type I nuclear receptors to be co-recruited after dual agonist treatment. We focused on ERα/glucocorticoid receptor interplay and demonstrated that it requires steroid receptor coactivators (SRC-2, SRC-3) and the mediator component MED14. We then validated this cooperative interplay on endogenous target genes in breast cancer cells. Taken together, this work highlights another layer of mechanistic complexity through which NRs cross-talk with each other on chromatin under multiple hormonal stimuli.


Assuntos
Receptor alfa de Estrogênio/metabolismo , Coativadores de Receptor Nuclear/metabolismo , Receptores de Glucocorticoides/metabolismo , Transcrição Gênica , Linhagem Celular Tumoral , Cromatina/metabolismo , Receptor alfa de Estrogênio/agonistas , Receptor alfa de Estrogênio/química , Genes Reporter , Células HeLa , Humanos , Complexo Mediador/metabolismo , Coativador 2 de Receptor Nuclear/metabolismo , Coativador 3 de Receptor Nuclear/metabolismo , Prolactina/genética , Estrutura Secundária de Proteína , Receptores de Glucocorticoides/agonistas
8.
J Biol Chem ; 287(4): 2579-90, 2012 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-22128152

RESUMO

Bacterial elongation factor P (EF-P) is the ortholog of archaeal and eukaryotic initiation factor 5A (eIF5A). EF-P shares sequence homology and crystal structure with eIF5A, but unlike eIF5A, EF-P does not undergo hypusine modification. Recently, two bacterial genes, yjeA and yjeK, encoding truncated homologs of class II lysyl-tRNA synthetase and of lysine-2,3-aminomutase, respectively, have been implicated in the modification of EF-P to convert a specific lysine to a hypothetical ß-lysyl-lysine. Here we present biochemical evidence for ß-lysyl-lysine modification in Escherichia coli EF-P and for its role in EF-P activity by characterizing native and recombinant EF-P proteins for their modification status and activity in vitro. Mass spectrometric analyses confirmed the lysyl modification at lysine 34 in native and recombinant EF-P proteins. The ß-lysyl-lysine isopeptide was identified in the exhaustive Pronase digests of native EF-P and recombinant EF-P isolated from E. coli coexpressing EF-P, YjeA, and YjeK but not in the digests of proteins derived from the vectors encoding EF-P alone or EF-P together with YjeA, indicating that both enzymes, YjeA and YjeK, are required for ß-lysylation of EF-P. Endogenous EF-P as well as the recombinant EF-P preparation containing ß-lysyl-EF-P stimulated N-formyl-methionyl-puromycin synthesis ∼4-fold over the preparations containing unmodified EF-P and/or α-lysyl-EF-P. The mutant lacking the modification site lysine (K34A) was inactive. This is the first report of biochemical evidence for the ß-lysylation of EF-P in vivo and the requirement for this modification for the activity of EF-P.


Assuntos
Desoxirribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Lisina/metabolismo , Fatores de Alongamento de Peptídeos/metabolismo , Processamento de Proteína Pós-Traducional/fisiologia , Desoxirribonucleases/química , Desoxirribonucleases/genética , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Lisina/química , Lisina/genética , Espectrometria de Massas , Fatores de Alongamento de Peptídeos/química , Fatores de Alongamento de Peptídeos/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
9.
Appl Environ Microbiol ; 77(12): 4223-5, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21531830

RESUMO

The group II azoreductase BTI1 utilizes NADPH to directly cleave azo bonds in water-soluble azo dyes, including quenchers of fluorescence. Unexpectedly, optimal reduction was dye specific, ranging from a pH of <5.5 for Janus green B, to pH 6.0 for methyl red, methyl orange, and BHQ-10, to pH >8.3 for flame orange.


Assuntos
Compostos Azo/metabolismo , NADH NADPH Oxirredutases/metabolismo , NADP/metabolismo , Concentração de Íons de Hidrogênio , Nitrorredutases , Especificidade por Substrato
10.
Elife ; 92020 10 27.
Artigo em Inglês | MEDLINE | ID: mdl-33108271

RESUMO

Cell cycle is a cellular process that is subject to stringent control. In contrast to the wealth of knowledge of proteins controlling the cell cycle, very little is known about the molecular role of lncRNAs (long noncoding RNAs) in cell-cycle progression. By performing genome-wide transcriptome analyses in cell-cycle-synchronized cells, we observed cell-cycle phase-specific induction of >2000 lncRNAs. Further, we demonstrate that an S-phase-upregulated lncRNA, SUNO1, facilitates cell-cycle progression by promoting YAP1-mediated gene expression. SUNO1 facilitates the cell-cycle-specific transcription of WTIP, a positive regulator of YAP1, by promoting the co-activator, DDX5-mediated stabilization of RNA polymerase II on chromatin. Finally, elevated SUNO1 levels are associated with poor cancer prognosis and tumorigenicity, implying its pro-survival role. Thus, we demonstrate the role of a S-phase up-regulated lncRNA in cell-cycle progression via modulating the expression of genes controlling cell proliferation.


Assuntos
Proliferação de Células/genética , Proteínas Correpressoras/genética , Proteínas do Citoesqueleto/genética , RNA Helicases DEAD-box/genética , Regulação da Expressão Gênica , RNA Longo não Codificante/genética , Transdução de Sinais/fisiologia , Proteínas Correpressoras/metabolismo , Proteínas do Citoesqueleto/metabolismo , RNA Helicases DEAD-box/metabolismo , Células HCT116 , Células HeLa , Humanos , RNA Longo não Codificante/metabolismo , Fase S , Regulação para Cima
11.
FEBS J ; 275(1): 44-58, 2008 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-18067580

RESUMO

The eukaryotic translation initiation factor 5A (eIF5A) is the only protein that contains hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine], which is required for its activity. Hypusine is formed by post-translational modification of one specific lysine (Lys50 for human eIF5A) by deoxyhypusine synthase and deoxyhypusine hydroxylase. To investigate the features of eIF5A required for its activity, we generated 49 mutations in human eIF5A-1, with a single amino acid substitution at the highly conserved residues or with N-terminal or C-terminal truncations, and tested mutant proteins in complementing the growth of a Saccharomyces cerevisiae eIF5A null strain. Growth-supporting activity was abolished in only a few mutant eIF5As (K47D, G49A, K50A, K50D, K50I, K50R, G52A and K55A), with substitutions at or near the hypusine modification site or with truncation of 21 amino acids from either the N-terminus or C-terminus. The inactivity of the Lys50 substitution proteins is obviously due to lack of deoxyhypusine modification. In contrast, K47D and G49A were effective substrates for deoxyhypusine synthase, yet failed to support growth, suggesting critical roles of Lys47 and Gly49 in eIF5A activity, possibly in its interaction with effector(s). By use of a UBHY-R strain harboring genetically engineered unstable eIF5A, we present evidence for the primary function of eIF5A in protein synthesis. When selected eIF5A mutant proteins were tested for their activity in protein synthesis, a close correlation was observed between their ability to enhance protein synthesis and growth, lending further support for a central role of eIF5A in translation.


Assuntos
Lisina/análogos & derivados , Fatores de Iniciação de Peptídeos/genética , Fatores de Iniciação de Peptídeos/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Sequência de Aminoácidos , Humanos , Lisina/química , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fatores de Iniciação de Peptídeos/química , Modificação Traducional de Proteínas , Proteínas de Ligação a RNA/química , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Fator de Iniciação de Tradução Eucariótico 5A
12.
Genome Biol ; 19(1): 46, 2018 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-29598823

RESUMO

BACKGROUND: The multifaceted control of gene expression requires tight coordination of regulatory mechanisms at transcriptional and post-transcriptional level. Here, we studied the interdependence of transcription initiation, splicing and polyadenylation events on single mRNA molecules by full-length mRNA sequencing. RESULTS: In MCF-7 breast cancer cells, we find 2700 genes with interdependent alternative transcription initiation, splicing and polyadenylation events, both in proximal and distant parts of mRNA molecules, including examples of coupling between transcription start sites and polyadenylation sites. The analysis of three human primary tissues (brain, heart and liver) reveals similar patterns of interdependency between transcription initiation and mRNA processing events. We predict thousands of novel open reading frames from full-length mRNA sequences and obtained evidence for their translation by shotgun proteomics. The mapping database rescues 358 previously unassigned peptides and improves the assignment of others. By recognizing sample-specific amino-acid changes and novel splicing patterns, full-length mRNA sequencing improves proteogenomics analysis of MCF-7 cells. CONCLUSIONS: Our findings demonstrate that our understanding of transcriptome complexity is far from complete and provides a basis to reveal largely unresolved mechanisms that coordinate transcription initiation and mRNA processing.


Assuntos
Poliadenilação , Splicing de RNA , RNA Mensageiro/metabolismo , Iniciação da Transcrição Genética , Humanos , Células MCF-7 , Motivos de Nucleotídeos , Poli A/metabolismo , Proteoma/genética , RNA Mensageiro/química , Proteínas de Ligação a RNA/metabolismo , Análise de Sequência de RNA , Transcriptoma
13.
Pediatr Hematol Oncol ; 24(4): 237-43, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17613866

RESUMO

Hypertransfusional (>8 transfusions/year) iron in liver biopsies collected immediately after transfusions in beta-thalassemia and sickle cell disease correlated with increased expression (RNA) for iron regulatory proteins 1 and 2 (3-, 9- to 11-fold) and hepcidin RNA: (5- to 8-fold) (each p <.01), while ferritin H and L RNA remained constant. A different H:L ferritin ratio in RNA (0.03) and protein (0.2-0.6) indicated disease-specific trends and suggests novel post-transcriptional effects. Increased iron regulatory proteins could stabilize the transferrin receptor mRNA and, thereby, iron uptake. Increased hepcidin, after correction of anemia by transfusion, likely reflects excess liver iron. Finally, the absence of a detectable change in ferritin mRNA indicates insufficient oxidative stress to significantly activate MARE/ARE promoters.


Assuntos
Anemia Falciforme/complicações , Peptídeos Catiônicos Antimicrobianos/genética , Ferritinas/genética , Homeostase/genética , Sobrecarga de Ferro/metabolismo , Proteínas Reguladoras de Ferro/genética , Ferro/metabolismo , Talassemia beta/complicações , Regulação da Expressão Gênica , Hepcidinas , Humanos , Sobrecarga de Ferro/etiologia , Proteína 1 Reguladora do Ferro/genética , Proteína 2 Reguladora do Ferro/genética , Estresse Oxidativo , RNA Mensageiro/análise , Reação Transfusional
14.
FEBS J ; 273(6): 1102-14, 2006 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-16519677

RESUMO

Eukaryotic translation initiation factor 5A (eIF5A) is the only cellular protein that contains the unusual amino acid hypusine [N(epsilon)-(4-amino-2-hydroxybutyl)lysine]. Vertebrates carry two genes that encode two eIF5A isoforms, eIF5A-1 and eIF5A-2, which, in humans, are 84% identical. eIF5A-1 mRNA (1.3 kb) and protein (18 kDa) are constitutively expressed in human cells. In contrast, expression of eIF5A-2 mRNA (0.7-5.6 kb) and eIF5A-2 protein (20 kDa) varies widely. Whereas eIF5A-2 mRNA was demonstrable in most cells, eIF5A-2 protein was detectable only in the colorectal and ovarian cancer-derived cell lines SW-480 and UACC-1598, which showed high overexpression of eIF5A-2 mRNA. Multiple forms of eIF5A-2 mRNA (5.6, 3.8, 1.6 and 0.7 kb) were identified as the products of one gene with various lengths of 3'-UTR, resulting from the use of different polyadenylation (AAUAAA) signals. The eIF5A-1 and eIF5A-2 precursor proteins were modified comparably in UACC-1598 cells and both were similarly stable. When eIF5A-1 and eIF5A-2 coding sequences were expressed from mammalian vectors in 293T cells, eIF5A-2 precursor was synthesized at a level comparable to that of eIF5A-1 precursor, indicating that the elements causing inefficient translation of eIF5A-2 mRNA reside outside of the open reading frame. On sucrose gradient separation of cytoplasmic RNA, only a small portion of total eIF5A-2 mRNA was associated with the polysomal fraction, compared with a much larger portion of eIF5A-1 mRNA in the polysomes. These findings suggest that the failure to detect eIF5A-2 protein even in eIF5A-2 mRNA positive cells is, at least in part, due to inefficient translation.


Assuntos
Neoplasias/metabolismo , Fatores de Iniciação de Peptídeos/metabolismo , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas de Ligação a RNA/metabolismo , Sequência de Bases , Linhagem Celular , Linhagem Celular Tumoral , Humanos , Lisina/análogos & derivados , Lisina/biossíntese , Dados de Sequência Molecular , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Fatores de Iniciação de Peptídeos/genética , Poliadenilação , Polirribossomos , Biossíntese de Proteínas , Isoformas de Proteínas , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Transfecção , Fator de Iniciação de Tradução Eucariótico 5A
15.
Methods Mol Biol ; 1402: 119-134, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26721487

RESUMO

RNA fluorescence in situ hybridization (FISH), long an indispensable tool for the detection and localization of RNA, is becoming an increasingly important complement to other gene expression analysis methods. Especially important for long noncoding RNAs (lncRNAs), RNA FISH adds the ability to distinguish between primary and mature lncRNA transcripts and thus to segregate the site of synthesis from the site of action.We detail a streamlined RNA FISH protocol for the simultaneous imaging of multiple primary and mature mRNA and lncRNA gene products and RNA variants in fixed mammalian cells. The technique makes use of fluorescently pre-labeled, short DNA oligonucleotides (circa 20 nucleotides in length), pooled into sets of up to 48 individual probes. The overall binding of multiple oligonucleotides to the same RNA target results in fluorescent signals that reveal clusters of RNAs or single RNA molecules as punctate spots without the need for enzymatic signal amplification. Visualization of these punctate signals, through the use of wide-field fluorescence microscopy, enables the counting of single transcripts down to one copy per cell. Additionally, by using probe sets with spectrally distinct fluorophores, multiplex analysis of gene-specific RNAs, or RNA variants, can be achieved. The presented examples illustrate how this method can add temporospatial information between the transcription event and both the location and the endurance of the mature lncRNA. We also briefly discuss post-processing of images and spot counting to demonstrate the capabilities of this method for the statistical analysis of RNA molecules per cell. This information can be utilized to determine both overall gene expression levels and cell-to-cell gene expression variation.


Assuntos
Hibridização in Situ Fluorescente/métodos , RNA Longo não Codificante/genética , Animais , Adesão Celular , Linhagem Celular Tumoral , Expressão Gênica , Humanos , Microscopia de Fluorescência , Permeabilidade , RNA Longo não Codificante/análise , Fixação de Tecidos/métodos
16.
Front Immunol ; 6: 457, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26441962

RESUMO

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease characterized by the production of anti-nuclear antibodies. SLE is one of many autoimmune disorders that have a strong gender bias, with 70-90% of SLE patients being female. Several explanations have been postulated to account for the severity of autoimmune diseases in females, including hormonal, microbiota, and gene dosage differences. X-linked toll-like receptors (TLRs) have recently been implicated in disease progression in females. Our previous studies using the 564Igi mouse model of SLE on a Tlr7 and Tlr9 double knockout background showed that the presence of Tlr8 on both X chromosomes was required for the production of IgG autoantibodies, Ifn-I expression and granulopoiesis in females. Here, we show the results of our investigation into the role of Tlr8 expression in SLE pathogenesis in 564Igi females. Female mice have an increase in serum pathogenic anti-RNA IgG2a and IgG2b autoantibodies. 564Igi mice have also been shown to have an increase in neutrophils in vivo, which are major contributors to Ifn-α expression. Here, we show that neutrophils from C57BL/6 mice express Ifn-α in response to 564 immune complexes and TLR8 activation. Bone marrow-derived macrophages from 564Igi females have a significant increase in Tlr8 expression compared to male-derived cells, and RNA fluorescence in situ hybridization data suggest that Tlr8 may escape X-inactivation in female-derived macrophages. These results propose a model by which females may be more susceptible to SLE pathogenesis due to inefficient inactivation of Tlr8.

17.
Methods Mol Biol ; 1211: 189-99, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25218386

RESUMO

RNA fluorescence in situ hybridization (FISH) has long been an indispensable tool for the detection and localization of RNA and is increasingly becoming an important complement to other gene expression analysis methods. We detail a streamlined RNA FISH protocol for the simultaneous imaging of multiple RNA gene products and RNA variants in fixed mammalian cells. The technique utilizes fluorescently pre-labeled, short DNA oligonucleotides (20 nucleotides in length), pooled into sets of up to 48 individual probes. The overall binding of multiple oligonucleotides to the same RNA target results in punctate fluorescent signals representing individual RNA molecules without the need for enzymatic signal amplification. Visualization of these punctate signals, through the use of wide-field fluorescence microscopy, enables the quantification of single RNA transcripts. Additionally, by utilizing probe sets with spectrally distinct fluorophores, multiplex analysis of specific RNAs, or RNA variants, can be achieved. We focus on the detection of a cytoplasmic mRNA and a nuclear long noncoding RNA to illustrate the benefits of this method for cell-specific detection and subcellular localization. Post-processing of images and spot counting is briefly discussed to demonstrate the capabilities of this method for the statistical analysis of RNA molecule number per cell, which is information that can be utilized to determine overall gene expression levels and cell-to-cell gene expression variation.


Assuntos
Núcleo Celular/ultraestrutura , Inibidor de Quinase Dependente de Ciclina p21/genética , Citoplasma/ultraestrutura , Hibridização in Situ Fluorescente/métodos , RNA Longo não Codificante/análise , RNA Mensageiro/análise , Adesão Celular , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Núcleo Celular/genética , Citoplasma/metabolismo , Humanos , Microscopia de Fluorescência/métodos , RNA Mensageiro/genética
18.
Eur J Biochem ; 270(21): 4254-63, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14622290

RESUMO

The phylogenetically conserved eukaryotic translation initiation factor 5A (eIF5A) is the only known cellular protein to contain the post-translationally derived amino acid hypusine [Nepsilon-(4-amino-2-hydroxybutyl)lysine]. Both eIF5A and its hypusine modification are essential for sustained cell proliferation. Normally only one eIF5A protein is expressed in human cells. Recently, we identified a second human EIF5A gene that would encode an isoform (eIF5A-2) of 84% sequence identity. Overexpression of eIF5A-2 mRNA in certain human cancer cells, in contrast to weak normal expression limited to human testis and brain, suggests EIF5A2 as a potential oncogene. However, eIF5A-2 protein has not been described in human or mammalian cells heretofore. Here, we describe the identification of eIF5A-2 protein in human colorectal and ovarian cancer lines, SW-480 and UACC-1598, that overexpress eIF5A-2 mRNAs. Functional characterization of the human isoforms revealed that either human EIF5A gene can complement growth of a yeast strain in which the yeast EIF5A genes were disrupted. This indicates functional similarity of the human isoforms in yeast and suggests that eIF5A-2 has an important role in eukaryotic cell survival similar to that of the ubiquitous eIF5A-1. Detectable structural differences were also noted, including lack of immunological cross-reactivity, formation of different complexes with deoxyhypusine synthase, and Km values (1.5 +/- 0.2 vs. 8.3 +/- 1.4 microm for eIF5A-1 and -2, respectively) as substrates for deoxyhypusine synthase in vitro. These physical characteristics and distinct amino acid sequences in the C-terminal domain together with differences in gene expression patterns imply differentiated, tissue-specific functions of the eIF5A-2 isoform in the mammalian organism and in cancer.


Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Linhagem Celular Tumoral , Clonagem Molecular , Primers do DNA , DNA Complementar , Ensaio de Desvio de Mobilidade Eletroforética , Feminino , Humanos , Dados de Sequência Molecular , Fatores de Iniciação de Peptídeos/química , Fatores de Iniciação de Peptídeos/genética , Processamento Pós-Transcricional do RNA , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Saccharomyces cerevisiae/genética , Homologia de Sequência de Aminoácidos
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