Your browser doesn't support javascript.
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 314
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
PLoS Genet ; 15(7): e1008240, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31365523

RESUMO

The RNA helicase SUV3 and the polynucleotide phosphorylase PNPase are involved in the degradation of mitochondrial mRNAs but their roles in vivo are not fully understood. Additionally, upstream processes, such as transcript maturation, have been linked to some of these factors, suggesting either dual roles or tightly interconnected mechanisms of mitochondrial RNA metabolism. To get a better understanding of the turn-over of mitochondrial RNAs in vivo, we manipulated the mitochondrial mRNA degrading complex in Drosophila melanogaster models and studied the molecular consequences. Additionally, we investigated if and how these factors interact with the mitochondrial poly(A) polymerase, MTPAP, as well as with the mitochondrial mRNA stabilising factor, LRPPRC. Our results demonstrate a tight interdependency of mitochondrial mRNA stability, polyadenylation and the removal of antisense RNA. Furthermore, disruption of degradation, as well as polyadenylation, leads to the accumulation of double-stranded RNAs, and their escape out into the cytoplasm is associated with an altered immune-response in flies. Together our results suggest a highly organised and inter-dependable regulation of mitochondrial RNA metabolism with far reaching consequences on cellular physiology.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , RNA Mitocondrial/química , RNA Mitocondrial/metabolismo , Animais , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , RNA Polimerases Dirigidas por DNA/genética , RNA Polimerases Dirigidas por DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/metabolismo , Feminino , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Poliadenilação , Polirribonucleotídeo Nucleotidiltransferase/genética , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , Estabilidade de RNA , RNA Antissenso/química , RNA Antissenso/metabolismo , RNA de Cadeia Dupla/química , RNA de Cadeia Dupla/metabolismo
2.
Methods Mol Biol ; 1927: 23-35, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30788783

RESUMO

Antisense RNA (asRNA) technology is an important tool for downregulating gene expression. When applying this strategy, the asRNA interference efficiency is determined by several elements including scaffold design, loop size, and relative abundance. Here, we take the Escherichia coli gene fabD encoding malonyl-CoA-[acyl-carrier-protein] transacylase as an example to describe the asRNA design with reliable and controllable interference efficiency. Real-time PCR and fluorescence assay methods are introduced to detect the interference efficiency at RNA level and protein level, respectively.


Assuntos
Regulação da Expressão Gênica , RNA Antissenso/genética , Proteína de Transporte de Acila S-Maloniltransferase/genética , Regulação para Baixo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ácido Graxo Sintase Tipo II/genética , Regulação Bacteriana da Expressão Gênica , Conformação de Ácido Nucleico , Plasmídeos/genética , Interferência de RNA , RNA Antissenso/química , Reação em Cadeia da Polimerase em Tempo Real/métodos
3.
ACS Nano ; 13(1): 706-717, 2019 01 22.
Artigo em Inglês | MEDLINE | ID: mdl-30543397

RESUMO

Displaying the advantage of nanoparticles in cancer targeting and drug delivery, micelles have shown great potential in cancer therapy. The mechanism for micelle targeting to cancer without the need for ligands is due to the size advantage of micelles within the lower end of the nanometer scale that is the optimal size for favoring the enhanced permeability and retention (EPR) effect while escaping trapping by macrophages. MicroRNAs are ubiquitous and play critical roles in regulating gene expression, cell growth, and cancer development. However, their in vivo delivery in medical applications is still challenging. Here, we report the targeted delivery of anti-miRNA to cancers via RNA micelles. The phi29 packaging RNA three-way junction (pRNA-3WJ) was used as a scaffold to construct micelles. An oligo with 8nt locked nucleic acid (LNA) complementary to the seed region of microRNA21(miR21) was included in the micelles as an interference molecule for cancer inhibition. These RNA micelles carrying anti-miR21 exhibited strong binding and internalization to cancer cells, inhibited the function of oncogenic miR21, enhanced the expression of the pro-apoptotic factor, and induced cell apoptosis. Animal trials revealed effective tumor targeting and inhibition in xenograft models. The inclusion of folate as a targeting ligand in the micelles did not show significant improvement of the therapeutic efficacy in vivo, suggesting that micelles can carry therapeutics to a target tumor and inhibit its growth without ligands.


Assuntos
Técnicas de Transferência de Genes , MicroRNAs/genética , Nanopartículas/química , Neoplasias Experimentais/terapia , RNA Antissenso/genética , Terapêutica com RNAi/métodos , Animais , Feminino , Células HT29 , Humanos , Camundongos , MicroRNAs/química , RNA Antissenso/química , Proteínas Virais/química
4.
Tissue Eng Part A ; 25(1-2): 12-23, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-29415631

RESUMO

Silk-based bioresorbable medical devices, such as screws, plates, and rods, have been under investigation due to their promising properties for orthopedic repairs. Options to functionalize these new devices for enhanced control of bone regeneration would also exploit the compatible processing methods used to generate the devices. MicroRNAs are important regulators of bone maintenance and formation, and miRNA-based therapeutics have the potential to aid bone repair, utilizing a transient therapeutic approach with local bioactivity. We hypothesized that silk-based orthopedic devices could be used for the local delivery of miRNAs, using anti-sense miR-214 (AS-miR-214), to inhibit endogenous expression of osteoinductive antagonist and thereby supporting the upregulation of osteoinductive target molecules activating transcription factor 4 (ATF4) and Osterix (Osx). AS-miR-214 silk devices, prepared using surface coating, demonstrated continuous release of miRNA inhibitors up to 7 days in vitro. Additionally, human mesenchymal stem cells seeded on AS-miR-214 silk films expressed higher levels of osteogenic genes ATF4, Osx, Runx2, and Osteocalcin. Interestingly, these cells exhibited lower cell viability and DNA content over 21 days. Conversely, the cells demonstrated significantly higher levels of alkaline phosphatase expression and calcium deposition compared with cells seeded on silk films with nontargeting miRNA controls. The study demonstrated that the silk-based orthopedic devices, in conjunction with bioactive miRNA-based therapeutics, may serve as a novel system for localized bone tissue engineering, enhancing osteogenesis at the implant interface while avoiding detrimental systematic side effects.


Assuntos
Materiais Biocompatíveis , Regeneração Óssea/efeitos dos fármacos , Células-Tronco Mesenquimais/metabolismo , Osteogênese/efeitos dos fármacos , RNA Antissenso , Seda , Engenharia Tecidual/métodos , Materiais Biocompatíveis/química , Materiais Biocompatíveis/farmacologia , Sobrevivência Celular , Células Endoteliais da Veia Umbilical Humana , Humanos , MicroRNAs/antagonistas & inibidores , MicroRNAs/metabolismo , Ortopedia , RNA Antissenso/química , RNA Antissenso/farmacologia , Seda/química , Seda/farmacologia
5.
FEMS Microbiol Lett ; 365(24)2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30376063

RESUMO

Recently, it has been found that bacteria secrete short RNAs able to affect gene expression in eukaryotic cells, while certain mammalian microRNAs shape the gut microbiome altering bacterial transcriptome. The involvement of bacterial RNAs in communication with other bacteria is also expected, but has not been documented yet. Here, we compared the fractions of extremely short (12-22 nucleotides) RNAs secreted by Escherichia coli grown in a pure culture and jointly with bacteria of the Paenibacillus genus. Besides fragments of rRNAs and tRNAs, abundant in all samples, secreted oligonucleotides (exoRNAs) predominantly contained GC-rich fragments of messenger and antisense RNAs processed from regions with stable secondary structures. They differed in composition from oligonucleotides of intracellular fraction, where fragments of small regulatory RNAs were prevalent. Both fractions contained RNAs capable of forming complementary duplexes, while for exoRNA samples a higher percentage of 3΄-end modified RNAs and different endonuclease cleavage were detected. The presence of a cohabiting bacterium altered the spectrum of E. coli exoRNAs, indicating a population-dependent control over their composition. Possible mechanisms of this effect are discussed.


Assuntos
Escherichia coli/metabolismo , RNA Antissenso/metabolismo , RNA Bacteriano/metabolismo , RNA Mensageiro/metabolismo , Transporte Biológico , Escherichia coli/química , Escherichia coli/genética , Genoma Bacteriano , Conformação de Ácido Nucleico , RNA Antissenso/química , RNA Antissenso/genética , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Ribossômico/química , RNA Ribossômico/genética , RNA Ribossômico/metabolismo , RNA de Transferência/química , RNA de Transferência/genética , RNA de Transferência/metabolismo
6.
Int J Mol Sci ; 19(1)2018 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-29301303

RESUMO

Natural antisense transcripts are RNA sequences that can be transcribed from both DNA strands at the same locus but in the opposite direction from the gene transcript. Because strand-specific high-throughput sequencing of the antisense transcriptome has only been available for less than a decade, many natural antisense transcripts were first described as long non-coding RNAs. Although the precise biological roles of natural antisense transcripts are not known yet, an increasing number of studies report their implication in gene expression regulation. Their expression levels are altered in many physiological and pathological conditions, including breast cancers. Among the potential clinical utilities of the natural antisense transcripts, the non-coding|coding transcript pairs are of high interest for treatment. Indeed, these pairs can be targeted by antisense oligonucleotides to specifically tune the expression of the coding-gene. Here, we describe the current knowledge about natural antisense transcripts, their varying molecular mechanisms as gene expression regulators, and their potential as prognostic or predictive biomarkers in breast cancers.


Assuntos
Neoplasias da Mama/genética , RNA Antissenso/metabolismo , Biomarcadores Tumorais/metabolismo , Feminino , Humanos , Modelos Genéticos , RNA Antissenso/química , Transcrição Genética
7.
Sci Rep ; 7(1): 12532, 2017 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-28970564

RESUMO

This study focused on determining design rules for gapmer-type antisense oligonucleotides (ASOs), that can differentiate cleavability of two SNP variants of RNA in the presence of ribonuclease H based on the mismatch type and position in the heteroduplex. We describe the influence of structural motifs formed by several arrangements of multiple mismatches (various types of mismatches and their position within the ASO/target RNA duplex) on RNase H cleavage selectivity of five different SNP types. The targets were mRNA fragments of APP, SCA3, SNCA and SOD1 genes, carrying C-to-G, G-to-C, G-to-A, A-to-G and C-to-U substitutions. The results show that certain arrangements of mismatches enhance discrimination between wild type and mutant SNP alleles of RNA in vitro as well as in HeLa cells. Among the over 120 gapmers tested, we found two gapmers that caused preferential degradation of the mutant allele APP 692 G and one that led to preferential cleavage of the mutant SNCA 53 A allele, both in vitro and in cells. However, several gapmers promoted selective cleavage of mRNA mutant alleles in in vitro experiments only.


Assuntos
Reparo de Erro de Pareamento de DNA/genética , Nucleotídeos/genética , RNA Antissenso/genética , Ribonuclease H/genética , Alelos , Células HeLa , Humanos , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/genética , Nucleotídeos/química , Polimorfismo de Nucleotídeo Único/genética , RNA Antissenso/química , RNA Mensageiro/genética , Ribonuclease H/química
9.
G3 (Bethesda) ; 7(5): 1607-1615, 2017 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-28364038

RESUMO

The 3' end of the small ribosomal RNAs (ssu rRNA) in bacteria is directly involved in the selection and binding of mRNA transcripts during translation initiation via well-documented interactions between a Shine-Dalgarno (SD) sequence located upstream of the initiation codon and an anti-SD (aSD) sequence at the 3' end of the ssu rRNA. Consequently, the 3' end of ssu rRNA (3'TAIL) is strongly conserved among bacterial species because a change in the region may impact the translation of many protein-coding genes. Escherichia coli and Bacillus subtilis differ in their 3' ends of ssu rRNA, being GAUCACCUCCUUA3' in E. coli and GAUCACCUCCUUUCU3' or GAUCACCUCCUUUCUA3' in B. subtilis Such differences in 3'TAIL lead to species-specific SDs (designated SDEc for E. coli and SDBs for B. subtilis) that can form strong and well-positioned SD/aSD pairing in one species but not in the other. Selection mediated by the species-specific 3'TAIL is expected to favor SDBs against SDEc in B. subtilis, but favor SDEc against SDBs in E. coli Among well-positioned SDs, SDEc is used more in E. coli than in B. subtilis, and SDBs more in B. subtilis than in E. coli Highly expressed genes and genes of high translation efficiency tend to have longer SDs than lowly expressed genes and genes with low translation efficiency in both species, but more so in B. subtilis than in E. coli Both species overuse SDs matching the bolded part of the 3'TAIL shown above. The 3'TAIL difference contributes to the host specificity of phages.


Assuntos
Bacillus subtilis/genética , Escherichia coli/genética , Iniciação Traducional da Cadeia Peptídica , RNA Ribossômico/genética , Sequências Reguladoras de Ácido Ribonucleico , Pareamento de Bases , Sequência Conservada , Regulação Bacteriana da Expressão Gênica , RNA Antissenso/química , RNA Antissenso/genética , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Ribossômico/química
10.
Nucleic Acids Res ; 45(9): 5614-5624, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28387839

RESUMO

RNA transcriptional regulators are emerging as versatile components for genetic network construction. However, these regulators suffer from incomplete repression in their OFF state, making their dynamic range less than that of their protein counterparts. This incomplete repression causes expression leak, which impedes the construction of larger synthetic regulatory networks as leak propagation can interfere with desired network function. To address this, we demonstrate how naturally derived antisense RNA-mediated transcriptional regulators can be configured to regulate both transcription and translation in a single compact RNA mechanism that functions in Escherichia coli. Using in vivo gene expression assays, we show that a combination of transcriptional termination and ribosome binding site sequestration increases repression from 85% to 98%, or activation from 10-fold to over 900-fold, in response to cognate antisense RNAs. We also show that orthogonal repressive versions of this mechanism can be created through engineering minimal antisense RNAs. Finally, to demonstrate the utility of this mechanism, we use it to reduce network leak in an RNA-only cascade. We anticipate these regulators will find broad use as synthetic biology moves beyond parts engineering to the design and construction of more sophisticated regulatory networks.


Assuntos
Regulação da Expressão Gênica , Biossíntese de Proteínas/genética , RNA/genética , Transcrição Genética , Sequência de Bases , Redes Reguladoras de Genes , Engenharia Genética , Mutação/genética , Plasmídeos/metabolismo , RNA/química , RNA Antissenso/química , RNA Antissenso/genética
11.
Nucleic Acids Res ; 45(9): 5523-5538, 2017 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-28334800

RESUMO

Current approaches to design efficient antisense RNAs (asRNAs) rely primarily on a thermodynamic understanding of RNA-RNA interactions. However, these approaches depend on structure predictions and have limited accuracy, arguably due to overlooking important cellular environment factors. In this work, we develop a biophysical model to describe asRNA-RNA hybridization that incorporates in vivo factors using large-scale experimental hybridization data for three model RNAs: a group I intron, CsrB and a tRNA. A unique element of our model is the estimation of the availability of the target region to interact with a given asRNA using a differential entropic consideration of suboptimal structures. We showcase the utility of this model by evaluating its prediction capabilities in four additional RNAs: a group II intron, Spinach II, 2-MS2 binding domain and glgC 5΄ UTR. Additionally, we demonstrate the applicability of this approach to other bacterial species by predicting sRNA-mRNA binding regions in two newly discovered, though uncharacterized, regulatory RNAs.


Assuntos
Fenômenos Biofísicos , Biologia Computacional/métodos , Modelos Biológicos , Hibridização de Ácido Nucleico , RNA Antissenso/química , RNA Bacteriano/química , Sequência de Bases , Conformação de Ácido Nucleico , RNA Mensageiro/metabolismo , Análise de Regressão , Termodinâmica
12.
Curr Opin Genet Dev ; 44: 17-29, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28208060

RESUMO

More than 30 incurable neurological and neuromuscular diseases are caused by simple microsatellite expansions consisted of 3-6 nucleotides. These repeats can occur in non-coding regions and often result in a dominantly inherited disease phenotype that is characteristic of a toxic RNA gain-of-function. The expanded RNA adopts unusual secondary structures, sequesters various RNA binding proteins to form insoluble nuclear foci, and causes cellular defects at a multisystem level. Nuclear foci are dynamic in size, shape and colocalization of RNA binding proteins in different expansion diseases and tissue types. This review sets to provide new insights into the disease mechanisms of RNA toxicity and foci modulation, in light of recent advancement on bi-directional transcription, antisense RNA, repeat-associated non-ATG translation and beyond.


Assuntos
Doenças do Sistema Nervoso/genética , Doenças Neuromusculares/genética , RNA/genética , Expansão das Repetições de Trinucleotídeos/genética , Humanos , Repetições de Microssatélites/genética , Doenças do Sistema Nervoso/patologia , Doenças Neuromusculares/patologia , Conformação de Ácido Nucleico , RNA/química , RNA Antissenso/química , RNA Antissenso/genética , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/genética
13.
RNA Biol ; 14(10): 1374-1388, 2017 10 03.
Artigo em Inglês | MEDLINE | ID: mdl-28102759

RESUMO

The unstable (CTG·CAG)n trinucleotide repeat in the myotonic dystrophy type 1 (DM1) locus is bidirectionally transcribed from genes with terminal overlap. By transcription in the sense direction, the DMPK gene produces various alternatively spliced mRNAs with a (CUG)n repeat in their 3' UTR. Expression in opposite orientation reportedly yields (CAG)n-repeat containing RNA, but both structure and biologic significance of this antisense gene (DM1-AS) are largely unknown. Via a combinatorial approach of computational and experimental analyses of RNA from unaffected individuals and DM1 patients we discovered that DM1-AS spans >6 kb, contains alternative transcription start sites and uses alternative polyadenylation sites up- and downstream of the (CAG)n repeat. Moreover, its primary transcripts undergo alternative splicing, whereby the (CAG)n segment is removed as part of an intron. Thus, in patients a mixture of DM1-AS RNAs with and without expanded (CAG)n repeat are produced. DM1-AS expression appears upregulated in patients, but transcript abundance remains very low in all tissues analyzed. Our data suggest that DM1-AS transcripts belong to the class of long non-coding RNAs. These and other biologically relevant implications for how (CAG)n-expanded transcripts may contribute to DM1 pathology can now be explored experimentally.


Assuntos
Distrofia Miotônica/genética , Miotonina Proteína Quinase/genética , RNA Antissenso/genética , RNA Mensageiro/química , Expansão das Repetições de Trinucleotídeos , Regiões 3' não Traduzidas , Adolescente , Processamento Alternativo , Estudos de Casos e Controles , Linhagem Celular , Biologia Computacional/métodos , Humanos , Masculino , Miotonina Proteína Quinase/química , Fases de Leitura Aberta , Poliadenilação , RNA Antissenso/química , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Sítio de Iniciação de Transcrição , Regulação para Cima
14.
Cell Rep ; 16(12): 3087-3096, 2016 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-27653675

RESUMO

There is considerable debate about the functionality of long non-coding RNAs (lncRNAs). Lack of sequence conservation has been used to argue against functional relevance. We investigated antisense lncRNAs, called COOLAIR, at the A. thaliana FLC locus and experimentally determined their secondary structure. The major COOLAIR variants are highly structured, organized by exon. The distally polyadenylated transcript has a complex multi-domain structure, altered by a single non-coding SNP defining a functionally distinct A. thaliana FLC haplotype. The A. thaliana COOLAIR secondary structure was used to predict COOLAIR exons in evolutionarily divergent Brassicaceae species. These predictions were validated through chemical probing and cloning. Despite the relatively low nucleotide sequence identity, the structures, including multi-helix junctions, show remarkable evolutionary conservation. In a number of places, the structure is conserved through covariation of a non-contiguous DNA sequence. This structural conservation supports a functional role for COOLAIR transcripts rather than, or in addition to, antisense transcription.


Assuntos
Evolução Molecular , Estrutura Secundária de Proteína , RNA Antissenso/química , RNA Longo não Codificante/química , Arabidopsis , Proteínas de Arabidopsis/química , Brassicaceae
15.
Microbiology ; 162(11): 2005-2016, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-27590250

RESUMO

Regulation of the Neisseria gonorrhoeae pilE gene is ill-defined. In this study, post-transcriptional effects on expression were assessed. In silico analysis predicts the formation of three putative stable stem-loop structures with favourable free energies within the 5' untranslated region of the pilE message. Using quantitative reverse transcriptase PCR analyses, we show that each loop structure forms, with introduced destabilizing stem-loop mutations diminishing loop stability. Utilizing a series of pilE translational fusions, deletion of either loop 1 or loop 2 caused a significant reduction of pilE mRNA resulting in reduced expression of the reporter gene. Consequently, the formation of the loops apparently protects the pilE transcript from degradation. Putative loop 3 contains the pilE ribosomal binding site. Consequently, its formation may influence translation. Analysis of a small RNA transcriptome revealed an antisense RNA being produced upstream of the pilE promoter that is predicted to hybridize across the 5' untranslated region loops. Insertional mutants were created where the antisense RNA is not transcribed. In these mutants, pilE transcript levels are greatly diminished, with any residual message apparently not being translated. Complementation of these insertion mutants in trans with the antisense RNA gene facilitates pilE translation yielding a pilus + phenotype. Overall, this study demonstrates a complex relationship between loop-dependent transcript protection and antisense RNA in modulating pilE expression levels.


Assuntos
Regiões 5' não Traduzidas , Proteínas de Fímbrias/genética , Regulação Bacteriana da Expressão Gênica , Neisseria gonorrhoeae/metabolismo , RNA Antissenso/química , RNA Antissenso/metabolismo , RNA Bacteriano/química , RNA Bacteriano/metabolismo , Proteínas de Fímbrias/química , Proteínas de Fímbrias/metabolismo , Sequências Repetidas Invertidas , Neisseria gonorrhoeae/química , Neisseria gonorrhoeae/genética , Conformação de Ácido Nucleico , RNA Antissenso/genética , RNA Bacteriano/genética
16.
Exp Hematol ; 44(11): 991-1001, 2016 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-27576131

RESUMO

Gene modulation of human hematopoietic stem and progenitor cells (HSPCs) harbors great potential for therapeutic application of these cells and presents a versatile tool in basic research to enhance our understanding of HSPC biology. However, stable genetic modification might be adverse, particularly in clinical settings. Here, we review a broad range of approaches to transient, nonviral modulation of protein expression with a focus on RNA-based methods. We compare different delivery methods and describe the usefulness of RNA molecules for overexpression as well as downregulation of proteins in HSPCs.


Assuntos
Engenharia Celular , Expressão Gênica , Técnicas de Transferência de Genes , Células-Tronco Hematopoéticas/metabolismo , RNA/genética , Terapia Genética/métodos , Transplante de Células-Tronco Hematopoéticas , Humanos , MicroRNAs/química , MicroRNAs/genética , RNA/administração & dosagem , RNA/química , RNA Antissenso/química , RNA Mensageiro/química , RNA Mensageiro/genética , Transfecção/métodos
17.
RNA Biol ; 13(9): 783-98, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27315567

RESUMO

The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3'-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3'-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125-80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3'-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2'-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the Paramyxoviridae which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences.


Assuntos
Ebolavirus/genética , Ebolavirus/metabolismo , RNA Viral/genética , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Virais/metabolismo , Composição de Bases , Sequência de Bases , Ensaio de Desvio de Mobilidade Eletroforética , Genoma Viral , Doença pelo Vírus Ebola/virologia , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , RNA Antissenso/química , RNA Antissenso/genética , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Viral/química , Proteínas de Ligação a RNA/química , Deleção de Sequência , Especificidade por Substrato , Fatores de Transcrição/química , Transcrição Genética , Proteínas Virais/química , Replicação Viral
18.
Biochemistry ; 55(9): 1283-6, 2016 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-26878348

RESUMO

Expansion of a GGGGCC/CCCCGG repeat sequence in the first intron of the C9ORF72 gene is a leading cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In this combined disorder, called c9FTD/ALS, the expansion is bidirectionally transcribed into sense and antisense repeat RNA associated with disease. To better understand the role of C9ORF72 repeat RNA in molecular disease pathology, we determined crystal structures of a [(CCCCGG)3(CCCC)] model antisense repeat RNA to 1.47 Å resolution. The RNA structure was an A-form-like double helix composed of repeating and regularly spaced tandem C:C mismatch pairs that perturbed helical geometry and surface charge. Solution studies revealed a preference for A-form-like helical conformations as the repeat number increased. Results provide a structural starting point for rationalizing the contribution of repeat RNA to c9FTD/ALS molecular disease mechanisms and for developing molecules to target C9ORF72 repeat RNA as potential therapeutics.


Assuntos
Pareamento Incorreto de Bases/fisiologia , Proteínas/química , Proteínas/genética , RNA Antissenso/química , RNA Antissenso/genética , Esclerose Amiotrófica Lateral/genética , Proteína C9orf72 , Expansão das Repetições de DNA/fisiologia , Demência Frontotemporal/genética , Humanos , Estrutura Secundária de Proteína , Difração de Raios X
19.
Nucleic Acids Res ; 44(4): 1937-43, 2016 Feb 29.
Artigo em Inglês | MEDLINE | ID: mdl-26717983

RESUMO

PNA is a promising molecule for antisense therapy of trinucleotide repeat disorders. We present the first crystal structures of RNA-PNA duplexes. They contain CUG repeats, relevant to myotonic dystrophy type I, and CAG repeats associated with poly-glutamine diseases. We also report the first PNA-PNA duplex containing mismatches. A comparison of the PNA homoduplex and the PNA-RNA heteroduplexes reveals PNA's intrinsic structural properties, shedding light on its reported sequence selectivity or intolerance of mismatches when it interacts with nucleic acids. PNA has a much lower helical twist than RNA and the resulting duplex has an intermediate conformation. PNA retains its overall conformation while locally there is much disorder, especially peptide bond flipping. In addition to the Watson-Crick pairing, the structures contain interesting interactions between the RNA's phosphate groups and the Π electrons of the peptide bonds in PNA.


Assuntos
Ácidos Nucleicos Peptídicos/química , RNA Antissenso/genética , RNA/química , Expansão das Repetições de Trinucleotídeos/genética , Pareamento de Bases , Cristalografia por Raios X , Humanos , Distrofia Miotônica/genética , Distrofia Miotônica/terapia , Ácidos Nucleicos Peptídicos/genética , Ácidos Nucleicos Peptídicos/uso terapêutico , Peptídeos/genética , RNA/genética , RNA Antissenso/química , RNA Antissenso/uso terapêutico , Repetições de Trinucleotídeos/genética
20.
Nucleic Acids Res ; 44(6): 2898-908, 2016 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-26826711

RESUMO

The RNase P-mediated endonucleolytic cleavage plays a crucial role in the 3' end processing and cellular accumulation of MALAT1, a nuclear-retained long noncoding RNA that promotes malignancy. The regulation of this cleavage event is largely undetermined. Here we characterize a broadly expressed natural antisense transcript at the MALAT1 locus, designated as TALAM1, that positively regulates MALAT1 levels by promoting the 3' end cleavage and maturation of MALAT1 RNA. TALAM1 RNA preferentially localizes at the site of transcription, and also interacts with MALAT1 RNA. Depletion of TALAM1 leads to defects in the 3' end cleavage reaction and compromises cellular accumulation of MALAT1. Conversely, overexpression of TALAM1 facilitates the cleavage reaction in trans Interestingly, TALAM1 is also positively regulated by MALAT1 at the level of both transcription and RNA stability. Together, our data demonstrate a novel feed-forward positive regulatory loop that is established to maintain the high cellular levels of MALAT1, and also unravel the existence of sense-antisense mediated regulatory mechanism for cellular lncRNAs that display RNase P-mediated 3' end processing.


Assuntos
Núcleo Celular/metabolismo , RNA Antissenso/genética , RNA Longo não Codificante/antagonistas & inibidores , Sequência de Bases , Linhagem Celular Tumoral , Regulação da Expressão Gênica , Células HCT116 , Células HeLa , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Clivagem do RNA , Estabilidade de RNA , RNA Antissenso/química , RNA Antissenso/metabolismo , RNA Longo não Codificante/genética , RNA Longo não Codificante/metabolismo , Transdução de Sinais
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA