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1.
Nucleic Acids Res ; 50(17): 9689-9704, 2022 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-36107773

RESUMO

SERPINA1 mRNAs encode the protease inhibitor α-1-antitrypsin and are regulated through post-transcriptional mechanisms. α-1-antitrypsin deficiency leads to chronic obstructive pulmonary disease (COPD) and liver cirrhosis, and specific variants in the 5'-untranslated region (5'-UTR) are associated with COPD. The NM_000295.4 transcript is well expressed and translated in lung and blood and features an extended 5'-UTR that does not contain a competing upstream open reading frame (uORF). We show that the 5'-UTR of NM_000295.4 folds into a well-defined multi-helix structural domain. We systematically destabilized mRNA structure across the NM_000295.4 5'-UTR, and measured changes in (SHAPE quantified) RNA structure and cap-dependent translation relative to a native-sequence reporter. Surprisingly, despite destabilizing local RNA structure, most mutations either had no effect on or decreased translation. Most structure-destabilizing mutations retained native, global 5'-UTR structure. However, those mutations that disrupted the helix that anchors the 5'-UTR domain yielded three groups of non-native structures. Two of these non-native structure groups refolded to create a stable helix near the translation initiation site that decreases translation. Thus, in contrast to the conventional model that RNA structure in 5'-UTRs primarily inhibits translation, complex folding of the NM_000295.4 5'-UTR creates a translation-optimized message by promoting accessibility at the translation initiation site.


Assuntos
Biossíntese de Proteínas , Doença Pulmonar Obstrutiva Crônica , alfa 1-Antitripsina/genética , Regiões 5' não Traduzidas , Humanos , Inibidores de Proteases , Doença Pulmonar Obstrutiva Crônica/genética , RNA Mensageiro/metabolismo
2.
PLoS Genet ; 17(11): e1009912, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34784346

RESUMO

α1-anti-trypsin (A1AT), encoded by SERPINA1, is a neutrophil elastase inhibitor that controls the inflammatory response in the lung. Severe A1AT deficiency increases risk for Chronic Obstructive Pulmonary Disease (COPD), however, the role of A1AT in COPD in non-deficient individuals is not well known. We identify a 2.1-fold increase (p = 2.5x10-6) in the use of a distal poly-adenylation site in primary lung tissue RNA-seq in 82 COPD cases when compared to 64 controls and replicate this in an independent study of 376 COPD and 267 controls. This alternative polyadenylation event involves two sites, a proximal and distal site, 61 and 1683 nucleotides downstream of the A1AT stop codon. To characterize this event, we measured the distal ratio in human primary tissue short read RNA-seq data and corroborated our results with long read RNA-seq data. Integrating these results with 3' end RNA-seq and nanoluciferase reporter assay experiments we show that use of the distal site yields mRNA transcripts with over 50-fold decreased translation efficiency and A1AT expression. We identified seven RNA binding proteins using enhanced CrossLinking and ImmunoPrecipitation precipitation (eCLIP) with one or more binding sites in the SERPINA1 3' UTR. We combined these data with measurements of the distal ratio in shRNA knockdown experiments, nuclear and cytoplasmic fractionation, and chemical RNA structure probing. We identify Quaking Homolog (QKI) as a modulator of SERPINA1 mRNA translation and confirm the role of QKI in SERPINA1 translation with luciferase reporter assays. Analysis of single-cell RNA-seq showed differences in the distribution of the SERPINA1 distal ratio among hepatocytes, macrophages, αß-Tcells and plasma cells in the liver. Alveolar Type 1,2, dendritic cells and macrophages also vary in their distal ratio in the lung. Our work reveals a complex post-transcriptional mechanism that regulates alternative polyadenylation and A1AT expression in COPD.


Assuntos
Pulmão/metabolismo , Doença Pulmonar Obstrutiva Crônica/genética , alfa 1-Antitripsina/genética , Linhagem Celular , Códon de Terminação/genética , Regulação da Expressão Gênica/genética , Hepatócitos/metabolismo , Humanos , Fígado/metabolismo , Pulmão/patologia , Macrófagos/metabolismo , Poliadenilação/genética , Proteínas Secretadas Inibidoras de Proteinases/genética , Proteínas Secretadas Inibidoras de Proteinases/metabolismo , Doença Pulmonar Obstrutiva Crônica/patologia , RNA-Seq , Análise de Célula Única , Linfócitos T/metabolismo
3.
Am J Respir Crit Care Med ; 201(5): 540-554, 2020 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-31661293

RESUMO

Rationale: The role of PI (protease inhibitor) type Z heterozygotes and additional rare variant genotypes in the gene encoding alpha-1 antitrypsin, SERPINA1 (serpin peptidase inhibitor, clade A, member 1), in determining chronic obstructive pulmonary disease risk and severity is controversial.Objectives: To comprehensively evaluate the effects of rare SERPINA1 variants on lung function and emphysema phenotypes in subjects with significant tobacco smoke exposure using deep gene resequencing and alpha-1 antitrypsin concentrations.Methods: DNA samples from 1,693 non-Hispanic white individuals, 385 African Americans, and 90 Hispanics with ≥20 pack-years smoking were resequenced for the identification of rare variants (allele frequency < 0.05) in 16.9 kB of SERPINA1.Measurements and Main Results: White PI Z heterozygotes confirmed by sequencing (MZ; n = 74) had lower post-bronchodilator FEV1 (P = 0.007), FEV1/FVC (P = 0.003), and greater computed tomography-based emphysema (P = 0.02) compared with 1,411 white individuals without PI Z, S, or additional rare variants denoted as VR. PI Z-containing compound heterozygotes (ZS/ZVR; n = 7) had lower FEV1/FVC (P = 0.02) and forced expiratory flow, midexpiratory phase (P = 0.009). Nineteen white heterozygotes for five non-S/Z coding variants associated with lower alpha-1 antitrypsin had greater computed tomography-based emphysema compared with those without rare variants. In African Americans, a 5' untranslated region insertion (rs568223361) was associated with lower alpha-1 antitrypsin and functional small airway disease (P = 0.007).Conclusions: In this integrative deep sequencing study of SERPINA1 with alpha-1 antitrypsin concentrations in a heavy smoker and chronic obstructive pulmonary disease cohort, we confirmed the effects of PI Z heterozygote and compound heterozygote genotypes. We demonstrate the cumulative effects of multiple SERPINA1 variants on alpha-1 antitrypsin deficiency, lung function, and emphysema, thus significantly increasing the frequency of SERPINA1 variation associated with respiratory disease in at-risk smokers.


Assuntos
Pulmão/fisiopatologia , Doença Pulmonar Obstrutiva Crônica/genética , Enfisema Pulmonar/genética , Fumar/epidemiologia , alfa 1-Antitripsina/genética , Adulto , Negro ou Afro-Americano , Idoso , Idoso de 80 Anos ou mais , Feminino , Volume Expiratório Forçado , Genótipo , Heterozigoto , Hispânico ou Latino , Humanos , Focalização Isoelétrica , Masculino , Fluxo Máximo Médio Expiratório , Pessoa de Meia-Idade , Fenótipo , Polimorfismo Genético , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Doença Pulmonar Obstrutiva Crônica/fisiopatologia , Enfisema Pulmonar/epidemiologia , Enfisema Pulmonar/metabolismo , Enfisema Pulmonar/fisiopatologia , Tomografia Computadorizada por Raios X , Capacidade Vital , População Branca , alfa 1-Antitripsina/metabolismo
4.
RNA ; 24(4): 513-528, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29317542

RESUMO

The impact of inherited and somatic mutations on messenger RNA (mRNA) structure remains poorly understood. Recent technological advances that leverage next-generation sequencing to obtain experimental structure data, such as SHAPE-MaP, can reveal structural effects of mutations, especially when these data are incorporated into structure modeling. Here, we analyze the ability of SHAPE-MaP to detect the relatively subtle structural changes caused by single-nucleotide mutations. We find that allele-specific sorting greatly improved our detection ability. Thus, we used SHAPE-MaP with a novel combination of clone-free robotic mutagenesis and allele-specific sorting to perform a rapid, comprehensive survey of noncoding somatic and inherited riboSNitches in two cancer-associated mRNAs, TPT1 and LCP1 Using rigorous thermodynamic modeling of the Boltzmann suboptimal ensemble, we identified a subset of mutations that change TPT1 and LCP1 RNA structure, with approximately 14% of all variants identified as riboSNitches. To confirm that these in vitro structures were biologically relevant, we tested how dependent TPT1 and LCP1 mRNA structures were on their environments. We performed SHAPE-MaP on TPT1 and LCP1 mRNAs in the presence or absence of cellular proteins and found that both mRNAs have similar overall folds in all conditions. RiboSNitches identified within these mRNAs in vitro likely exist under biological conditions. Overall, these data reveal a robust mRNA structural landscape where differences in environmental conditions and most sequence variants do not significantly alter RNA structural ensembles. Finally, predicting riboSNitches in mRNAs from sequence alone remains particularly challenging; these data will provide the community with benchmarks for further algorithmic development.


Assuntos
Biomarcadores Tumorais/genética , Proteínas dos Microfilamentos/genética , Polimorfismo de Nucleotídeo Único/genética , Dobramento de RNA/fisiologia , RNA Mensageiro/genética , Alelos , Sequência de Bases , Linhagem Celular , Bases de Dados Genéticas , Variação Genética/genética , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , RNA Mensageiro/química , Proteínas de Ligação a RNA/genética , Termodinâmica , Proteína Tumoral 1 Controlada por Tradução
5.
Proc Natl Acad Sci U S A ; 114(47): E10244-E10253, 2017 11 21.
Artigo em Inglês | MEDLINE | ID: mdl-29109288

RESUMO

Chronic obstructive pulmonary disease (COPD) affects over 65 million individuals worldwide, where α-1-antitrypsin deficiency is a major genetic cause of the disease. The α-1-antitrypsin gene, SERPINA1, expresses an exceptional number of mRNA isoforms generated entirely by alternative splicing in the 5'-untranslated region (5'-UTR). Although all SERPINA1 mRNAs encode exactly the same protein, expression levels of the individual mRNAs vary substantially in different human tissues. We hypothesize that these transcripts behave unequally due to a posttranscriptional regulatory program governed by their distinct 5'-UTRs and that this regulation ultimately determines α-1-antitrypsin expression. Using whole-transcript selective 2'-hydroxyl acylation by primer extension (SHAPE) chemical probing, we show that splicing yields distinct local 5'-UTR secondary structures in SERPINA1 transcripts. Splicing in the 5'-UTR also changes the inclusion of long upstream ORFs (uORFs). We demonstrate that disrupting the uORFs results in markedly increased translation efficiencies in luciferase reporter assays. These uORF-dependent changes suggest that α-1-antitrypsin protein expression levels are controlled at the posttranscriptional level. A leaky-scanning model of translation based on Kozak translation initiation sequences alone does not adequately explain our quantitative expression data. However, when we incorporate the experimentally derived RNA structure data, the model accurately predicts translation efficiencies in reporter assays and improves α-1-antitrypsin expression prediction in primary human tissues. Our results reveal that RNA structure governs a complex posttranscriptional regulatory program of α-1-antitrypsin expression. Crucially, these findings describe a mechanism by which genetic alterations in noncoding gene regions may result in α-1-antitrypsin deficiency.


Assuntos
Processamento Alternativo/genética , Modelos Biológicos , Biossíntese de Proteínas/genética , RNA Mensageiro/química , alfa 1-Antitripsina/genética , Regiões 5' não Traduzidas/genética , Células A549 , Sequência de Bases , Células Hep G2 , Humanos , Mutagênese , Fases de Leitura Aberta/genética , Doença Pulmonar Obstrutiva Crônica/genética , Relação Quantitativa Estrutura-Atividade , Isoformas de RNA/química , Isoformas de RNA/genética , RNA Mensageiro/genética , Deficiência de alfa 1-Antitripsina/genética
6.
J Neurosci ; 38(39): 8407-8420, 2018 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-30150364

RESUMO

Previous studies have shown that common variants of the gene coding for FK506-binding protein 51 (FKBP5), a critical regulator of glucocorticoid sensitivity, affect vulnerability to stress-related disorders. In a previous report, FKBP5 rs1360780 was identified as a functional variant because of its effect on gene methylation. Here we report evidence for a novel functional FKBP5 allele, rs3800373. This study assessed the association between rs3800373 and post-traumatic chronic pain in 1607 women and men from two ethnically diverse human cohorts. The molecular mechanism through which rs3800373 affects adverse outcomes was established via in silico, in vivo, and in vitro analyses. The rs3800373 minor allele predicted worse adverse outcomes after trauma exposure, such that individuals with the minor (risk) allele developed more severe post-traumatic chronic musculoskeletal pain. Among these individuals, peritraumatic circulating FKBP5 expression levels increased as cortisol and glucocorticoid receptor (NR3C1) mRNA levels increased, consistent with increased glucocorticoid resistance. Bioinformatic, in vitro, and mutational analyses indicate that the rs3800373 minor allele reduces the binding of a stress- and pain-associated microRNA, miR-320a, to FKBP5 via altering the FKBP5 mRNA 3'UTR secondary structure (i.e., is a riboSNitch). This results in relatively greater FKBP5 translation, unchecked by miR-320a. Overall, these results identify an important gene-miRNA interaction influencing chronic pain risk in vulnerable individuals and suggest that exogenous methods to achieve targeted reduction in poststress FKBP5 mRNA expression may constitute useful therapeutic strategies.SIGNIFICANCE STATEMENTFKBP5 is a critical regulator of the stress response. Previous studies have shown that dysregulation of the expression of this gene plays a role in the pathogenesis of chronic pain development as well as a number of comorbid neuropsychiatric disorders. In the current study, we identified a functional allele (rs3800373) in the 3'UTR of FKBP5 that influences vulnerability to chronic post-traumatic pain in two ethnic cohorts. Using multiple complementary experimental approaches, we show that the FKBP5 rs3800373 minor allele alters the secondary structure of FKBP5 mRNA, decreasing the binding of a stress- and pain-associated microRNA, miR-320a. This results in relatively greater FKBP5 translation, unchecked by miR-320a, increasing glucocorticoid resistance and increasing vulnerability to post-traumatic pain.


Assuntos
Dor Crônica/genética , MicroRNAs/genética , Dor Musculoesquelética/genética , Proteínas de Ligação a Tacrolimo/genética , Regiões 3' não Traduzidas , Adulto , Negro ou Afro-Americano/genética , Alelos , Dor Crônica/metabolismo , Feminino , Genótipo , Humanos , Masculino , MicroRNAs/metabolismo , Dor Musculoesquelética/metabolismo , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Estrutura Secundária de Proteína , RNA Mensageiro/metabolismo , Receptores de Glucocorticoides/metabolismo , Proteínas de Ligação a Tacrolimo/metabolismo , População Branca/genética , Adulto Jovem
7.
Nature ; 494(7437): 366-70, 2013 Feb 21.
Artigo em Inglês | MEDLINE | ID: mdl-23389445

RESUMO

Several mutations are required for cancer development, and genome sequencing has revealed that many cancers, including breast cancer, have somatic mutation spectra dominated by C-to-T transitions. Most of these mutations occur at hydrolytically disfavoured non-methylated cytosines throughout the genome, and are sometimes clustered. Here we show that the DNA cytosine deaminase APOBEC3B is a probable source of these mutations. APOBEC3B messenger RNA is upregulated in most primary breast tumours and breast cancer cell lines. Tumours that express high levels of APOBEC3B have twice as many mutations as those that express low levels and are more likely to have mutations in TP53. Endogenous APOBEC3B protein is predominantly nuclear and the only detectable source of DNA C-to-U editing activity in breast cancer cell-line extracts. Knockdown experiments show that endogenous APOBEC3B correlates with increased levels of genomic uracil, increased mutation frequencies, and C-to-T transitions. Furthermore, induced APOBEC3B overexpression causes cell cycle deviations, cell death, DNA fragmentation, γ-H2AX accumulation and C-to-T mutations. Our data suggest a model in which APOBEC3B-catalysed deamination provides a chronic source of DNA damage in breast cancers that could select TP53 inactivation and explain how some tumours evolve rapidly and manifest heterogeneity.


Assuntos
Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Citidina Desaminase/metabolismo , Mutagênese , Mutação Puntual , Sequência de Bases , Biocatálise , Neoplasias da Mama/patologia , Morte Celular , Linhagem Celular Tumoral , Citidina Desaminase/genética , Dano ao DNA/genética , Fragmentação do DNA , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Desaminação , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Humanos , Antígenos de Histocompatibilidade Menor , Mutagênese/genética , Fenótipo , Mutação Puntual/genética , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Uracila/metabolismo
8.
Biophys J ; 113(2): 290-301, 2017 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-28625696

RESUMO

When a ribonucleic acid (RNA) molecule folds, it often does not adopt a single, well-defined conformation. The folding energy landscape of an RNA is highly dependent on its nucleotide sequence and molecular environment. Cellular molecules sometimes alter the energy landscape, thereby changing the ensemble of likely low-energy conformations. The effects of these energy landscape changes on the conformational ensemble are particularly challenging to visualize for large RNAs. We have created a robust approach for visualizing the conformational ensemble of RNAs that is well suited for in vitro versus in vivo comparisons. Our method creates a stable map of conformational space for a given RNA sequence. We first identify single point mutations in the RNA that maximally sample suboptimal conformational space based on the ensemble's partition function. Then, we cluster these diverse ensembles to identify the most diverse partition functions for Boltzmann stochastic sampling. By using, to our knowledge, a novel nestedness distance metric, we iteratively add mutant suboptimal ensembles to converge on a stable 2D map of conformational space. We then compute the selective 2' hydroxyl acylation by primer extension (SHAPE)-directed ensemble for the RNA folding under different conditions, and we project these ensembles on the map to visualize. To validate our approach, we established a conformational map of the Vibrio vulnificus add adenine riboswitch that reveals five classes of structures. In the presence of adenine, projection of the SHAPE-directed sampling correctly identified the on-conformation; without the ligand, only off-conformations were visualized. We also collected the whole-transcript in vitro and in vivo SHAPE-MaP for human ß-actin messenger RNA that revealed similar global folds in both conditions. Nonetheless, a comparison of in vitro and in vivo data revealed that specific regions exhibited significantly different SHAPE-MaP profiles indicative of structural rearrangements, including rearrangement consistent with binding of the zipcode protein in a region distal to the stop codon.


Assuntos
Conformação de Ácido Nucleico , RNA , Actinas/química , Actinas/genética , Actinas/metabolismo , Adenina/química , Adenina/metabolismo , Humanos , Modelos Genéticos , Modelos Moleculares , Mutação , RNA/química , RNA/metabolismo , Riboswitch/fisiologia , Processos Estocásticos , Termodinâmica , Vibrio vulnificus
9.
J Biol Chem ; 288(24): 17253-60, 2013 Jun 14.
Artigo em Inglês | MEDLINE | ID: mdl-23640892

RESUMO

APOBEC3A (A3A) is a myeloid lineage-specific DNA cytosine deaminase with a role in innate immunity to foreign DNA. Previous studies have shown that heterologously expressed A3A is genotoxic, suggesting that monocytes may have a mechanism to regulate this enzyme. Indeed, we observed no significant cytotoxicity when interferon was used to induce the expression of endogenous A3A in CD14(+)-enriched primary cells or the monocytic cell line THP-1. In contrast, doxycycline-induced A3A in HEK293 cells caused major cytotoxicity at protein levels lower than those observed when CD14(+) cells were stimulated with interferon. Immunofluorescent microscopy of interferon-stimulated CD14(+) and THP-1 cells revealed that endogenous A3A is cytoplasmic, in stark contrast to stably or transiently transfected A3A, which has a cell-wide localization. A3A constructs engineered to be cytoplasmic are also nontoxic in HEK293 cells. These data combine to suggest that monocytic cells use a cytoplasmic retention mechanism to control A3A and avert genotoxicity during innate immune responses.


Assuntos
Citidina Desaminase/fisiologia , Citoplasma/enzimologia , Dano ao DNA , Proteínas/fisiologia , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular , Expressão Gênica , Células HEK293 , Histonas/metabolismo , Humanos , Imunidade Inata , Receptores de Lipopolissacarídeos/metabolismo , Monócitos/enzimologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo
10.
J Biol Chem ; 287(41): 34801-8, 2012 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-22896697

RESUMO

Multiple studies have indicated that the TET oxidases and, more controversially, the activation-induced cytidine deaminase/APOBEC deaminases have the capacity to convert genomic DNA 5-methylcytosine (MeC) into altered nucleobases that provoke excision repair and culminate in the replacement of the original MeC with a normal cytosine (C). We show that human APOBEC3A (A3A) efficiently deaminates both MeC to thymine (T) and normal C to uracil (U) in single-stranded DNA substrates. In comparison, the related enzyme APOBEC3G (A3G) has undetectable MeC to T activity and 10-fold less C to U activity. Upon 100-fold induction of endogenous A3A by interferon, the MeC status of bulk chromosomal DNA is unaltered, whereas both MeC and C nucleobases in transfected plasmid DNA substrates are highly susceptible to editing. Knockdown experiments show that endogenous A3A is the source of both of these cellular DNA deaminase activities. This is the first evidence for nonchromosomal DNA MeC to T editing in human cells. These biochemical and cellular data combine to suggest a model in which the expanded substrate versatility of A3A may be an evolutionary adaptation that occurred to fortify its innate immune function in foreign DNA clearance by myeloid lineage cell types.


Assuntos
5-Metilcitosina/metabolismo , Citidina Desaminase/metabolismo , DNA/metabolismo , Imunidade Inata , Proteínas/metabolismo , 5-Metilcitosina/imunologia , Citidina Desaminase/imunologia , DNA/imunologia , Desaminação , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/imunologia , Células HEK293 , Humanos , Interferons/imunologia , Interferons/farmacologia , Plasmídeos/imunologia , Plasmídeos/farmacologia , Proteínas/imunologia , Timina/imunologia , Timina/metabolismo , Uracila/imunologia , Uracila/metabolismo
11.
Biosci Rep ; 43(3)2023 03 31.
Artigo em Inglês | MEDLINE | ID: mdl-36689327

RESUMO

RNA is a fundamental biomolecule that has many purposes within cells. Due to its single-stranded and flexible nature, RNA naturally folds into complex and dynamic structures. Recent technological and computational advances have produced an explosion of RNA structural data. Many RNA structures have regulatory and functional properties. Studying the structure of nascent RNAs is particularly challenging due to their low abundance and long length, but their structures are important because they can influence RNA processing. Precursor RNA processing is a nexus of pathways that determines mature isoform composition and that controls gene expression. In this review, we examine what is known about human nascent RNA structure and the influence of RNA structure on processing of precursor RNAs. These known structures provide examples of how other nascent RNAs may be structured and show how novel RNA structures may influence RNA processing including splicing and polyadenylation. RNA structures can be targeted therapeutically to treat disease.


Assuntos
Precursores de RNA , Splicing de RNA , Humanos , Precursores de RNA/genética , Precursores de RNA/metabolismo , RNA/genética , Poliadenilação , Expressão Gênica
12.
J Biol Chem ; 286(5): 3387-95, 2011 Feb 04.
Artigo em Inglês | MEDLINE | ID: mdl-21123176

RESUMO

APOBEC3G (A3G) is an antiviral protein that binds RNA and single-stranded DNA (ssDNA). The oligomerization state of A3G is likely to be influenced by these nucleic acid interactions. We applied the power of nanoimaging atomic force microscopy technology to characterize the role of ssDNA in A3G oligomerization. We used recombinant human A3G prepared from HEK-293 cells and specially designed DNA substrates that enable free A3G to be distinguished unambiguously from DNA-bound protein complexes. This DNA substrate can be likened to a molecular ruler because it consists of a 235-bp double-stranded DNA visual tag spliced to a 69-nucleotide ssDNA substrate. This hybrid substrate enabled us to use volume measurements to determine A3G stoichiometry in both free and ssDNA-bound states. We observed that free A3G is primarily monomeric, whereas ssDNA-complexed A3G is mostly dimeric. A3G stoichiometry increased slightly with the addition of Mg(2+), but dimers still predominated when Mg(2+) was depleted. A His-248/His-250 Zn(2+)-mediated intermolecular bridge was observed in a catalytic domain crystal structure (Protein Data Bank code 3IR2); however, atomic force microscopy analyses showed that the stoichiometry of the A3G-ssDNA complexes changed insignificantly when these residues were mutated to Ala. We conclude that A3G exchanges between oligomeric forms in solution with monomers predominating and that this equilibrium shifts toward dimerization upon binding ssDNA.


Assuntos
Citidina Desaminase/química , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/química , Microscopia de Força Atômica/métodos , Multimerização Proteica , Desaminase APOBEC-3G , Citidina Desaminase/metabolismo , Sondas de DNA/química , Humanos , Magnésio/farmacologia , Ligação Proteica , Proteínas de Ligação a RNA
13.
J Virol ; 85(21): 11220-34, 2011 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-21835787

RESUMO

Successful intracellular pathogens must evade or neutralize the innate immune defenses of their host cells and render the cellular environment permissive for replication. For example, to replicate efficiently in CD4(+) T lymphocytes, human immunodeficiency virus type 1 (HIV-1) encodes a protein called viral infectivity factor (Vif) that promotes pathogenesis by triggering the degradation of the retrovirus restriction factor APOBEC3G. Other APOBEC3 proteins have been implicated in HIV-1 restriction, but the relevant repertoire remains ambiguous. Here we present the first comprehensive analysis of the complete, seven-member human and rhesus APOBEC3 families in HIV-1 restriction. In addition to APOBEC3G, we find that three other human APOBEC3 proteins, APOBEC3D, APOBEC3F, and APOBEC3H, are all potent HIV-1 restriction factors. These four proteins are expressed in CD4(+) T lymphocytes, are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, mutate proviral DNA, and are counteracted by HIV-1 Vif. Furthermore, APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H of the rhesus macaque also are packaged into and restrict Vif-deficient HIV-1 when stably expressed in T cells, and they are all neutralized by the simian immunodeficiency virus Vif protein. On the other hand, neither human nor rhesus APOBEC3A, APOBEC3B, nor APOBEC3C had a significant impact on HIV-1 replication. These data strongly implicate a combination of four APOBEC3 proteins--APOBEC3D, APOBEC3F, APOBEC3G, and APOBEC3H--in HIV-1 restriction.


Assuntos
Citosina Desaminase/imunologia , HIV-1/imunologia , HIV-1/patogenicidade , Fatores de Virulência/deficiência , Produtos do Gene vif do Vírus da Imunodeficiência Humana/deficiência , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD4-Positivos/virologia , Células Cultivadas , Citosina Desaminase/metabolismo , Humanos , Macaca mulatta , Dados de Sequência Molecular , Análise de Sequência de DNA , Vírus da Imunodeficiência Símia/imunologia , Vírus da Imunodeficiência Símia/patogenicidade
14.
Elife ; 112022 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-35695373

RESUMO

Splicing is highly regulated and is modulated by numerous factors. Quantitative predictions for how a mutation will affect precursor mRNA (pre-mRNA) structure and downstream function are particularly challenging. Here, we use a novel chemical probing strategy to visualize endogenous precursor and mature MAPT mRNA structures in cells. We used these data to estimate Boltzmann suboptimal structural ensembles, which were then analyzed to predict consequences of mutations on pre-mRNA structure. Further analysis of recent cryo-EM structures of the spliceosome at different stages of the splicing cycle revealed that the footprint of the Bact complex with pre-mRNA best predicted alternative splicing outcomes for exon 10 inclusion of the alternatively spliced MAPT gene, achieving 74% accuracy. We further developed a ß-regression weighting framework that incorporates splice site strength, RNA structure, and exonic/intronic splicing regulatory elements capable of predicting, with 90% accuracy, the effects of 47 known and 6 newly discovered mutations on inclusion of exon 10 of MAPT. This combined experimental and computational framework represents a path forward for accurate prediction of splicing-related disease-causing variants.


Assuntos
Processamento Alternativo , Precursores de RNA , Éxons , Íntrons , Mutação , Precursores de RNA/genética , Precursores de RNA/metabolismo , Sítios de Splice de RNA , Splicing de RNA , RNA Mensageiro/genética
15.
Antibodies (Basel) ; 8(3)2019 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-31544853

RESUMO

The DNA cytosine deaminase APOBEC3B (A3B) is normally an antiviral factor in the innate immune response. However, A3B has been implicated in cancer mutagenesis, particularly in solid tumors of the bladder, breast, cervix, head/neck, and lung. Here, we report data on the generation and characterization of a rabbit monoclonal antibody (mAb) for human A3B. One mAb, 5210-87-13, demonstrates utility in multiple applications, including ELISA, immunoblot, immunofluorescence microscopy, and immunohistochemistry. In head-to-head tests with commercial reagents, 5210-87-13 was the only rabbit monoclonal suitable for detecting native A3B and for immunohistochemical quantification of A3B in tumor tissues. This novel mAb has the potential to enable a wide range of fundamental and clinical studies on A3B in human biology and disease.

16.
PLoS One ; 10(10): e0140885, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26480348

RESUMO

Genome-wide association studies aim to correlate genotype with phenotype. Many common diseases including Type II diabetes, Alzheimer's, Parkinson's and Chronic Obstructive Pulmonary Disease (COPD) are complex genetic traits with hundreds of different loci that are associated with varied disease risk. Identifying common features in the genes associated with each disease remains a challenge. Furthermore, the role of post-transcriptional regulation, and in particular alternative splicing, is still poorly understood in most multigenic diseases. We therefore compiled comprehensive lists of genes associated with Type II diabetes, Alzheimer's, Parkinson's and COPD in an attempt to identify common features of their corresponding mRNA transcripts within each gene set. The SERPINA1 gene is a well-recognized genetic risk factor of COPD and it produces 11 transcript variants, which is exceptional for a human gene. This led us to hypothesize that other genes associated with COPD, and complex disorders in general, are highly transcriptionally diverse. We found that COPD-associated genes have a statistically significant enrichment in transcript complexity stemming from a disproportionately high level of alternative splicing, however, Type II Diabetes, Alzheimer's and Parkinson's disease genes were not significantly enriched. We also identified a subset of transcriptionally complex COPD-associated genes (~40%) that are differentially expressed between mild, moderate and severe COPD. Although the genes associated with other lung diseases are not extensively documented, we found preliminary data that idiopathic pulmonary disease genes, but not cystic fibrosis modulators, are also more transcriptionally complex. Interestingly, complex COPD transcripts are more often the product of alternative acceptor site usage. To verify the biological importance of these alternative transcripts, we used RNA-sequencing analyses to determine that COPD-associated genes are frequently expressed in lung and liver tissues and are regulated in a tissue-specific manner. Additionally, many complex COPD-associated genes are spliced differently between COPD and non-COPD patients. Our analysis therefore suggests that post-transcriptional regulation, particularly alternative splicing, is an important feature specific to COPD disease etiology that warrants further investigation.


Assuntos
Biologia Computacional , Doença Pulmonar Obstrutiva Crônica/genética , Composição de Bases , Fibrose Cística/genética , Éxons/genética , Loci Gênicos/genética , Humanos , Fibrose Pulmonar Idiopática/genética , Splicing de RNA , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica , alfa 1-Antitripsina/genética
17.
Cell Cycle ; 12(5): 762-72, 2013 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-23388464

RESUMO

Humans have seven APOBEC3 DNA cytosine deaminases. The activity of these enzymes allows them to restrict a variety of retroviruses and retrotransposons, but may also cause pro-mutagenic genomic uracil lesions. During interphase the APOBEC3 proteins have different subcellular localizations: cell-wide, cytoplasmic or nuclear. This implies that only a subset of APOBEC3s have contact with nuclear DNA. However, during mitosis, the nuclear envelope breaks down and cytoplasmic proteins may enter what was formerly a privileged zone. To address the hypothesis that all APOBEC3 proteins have access to genomic DNA, we analyzed the localization of the APOBEC3 proteins during mitosis. We show that APOBEC3A, APOBEC3C and APOBEC3H are excluded from condensed chromosomes, but become cell-wide during telophase. However, APOBEC3B, APOBEC3D, APOBEC3F and APOBEC3G are excluded from chromatin throughout mitosis. After mitosis, APOBEC3B becomes nuclear, and APOBEC3D, APOBEC3F and APOBEC3G become cytoplasmic. Both structural motifs as well as size may be factors in regulating chromatin exclusion. Deaminase activity was not dependent on cell cycle phase. We also analyzed APOBEC3-induced cell cycle perturbations as a measure of each enzyme's capacity to inflict genomic DNA damage. AID, APOBEC3A and APOBEC3B altered the cell cycle profile, and, unexpectedly, APOBEC3D also caused changes. We conclude that several APOBEC3 family members have access to the nuclear compartment and can impede the cell cycle, most likely through DNA deamination and the ensuing DNA damage response. Such genomic damage may contribute to carcinogenesis, as demonstrated by AID in B cell cancers and, recently, APOBEC3B in breast cancers.


Assuntos
Citosina Desaminase/metabolismo , DNA/metabolismo , Genoma Humano/genética , Mitose , Cromossomos Humanos/metabolismo , Citosina Desaminase/química , Desaminação/genética , Proteínas de Fluorescência Verde/metabolismo , Células HEK293 , Células HeLa , Humanos , Interfase/genética , Estrutura Terciária de Proteína , Transporte Proteico/genética , Fase S/genética , Frações Subcelulares/enzimologia , Telófase/genética , Transfecção
18.
J Mol Biol ; 419(5): 301-14, 2012 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-22446380

RESUMO

Members of the APOBEC (apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like) protein family catalyze DNA cytosine deamination and underpin a variety of immune defenses. For instance, several family members, including APOBEC3B (A3B), elicit strong retrotransposon and retrovirus restriction activities. However, unlike the other proteins, A3B is the only family member with steady-state nuclear localization. Here, we show that A3B nuclear import is an active process requiring at least one amino acid (Val54) within an N-terminal motif analogous to the nuclear localization determinant of the antibody gene diversification enzyme AID (activation-induced cytosine deaminase). Mechanistic conservation with AID is further suggested by A3B's capacity to interact with the same subset of importin proteins. Despite these mechanistic similarities, enforced A3B expression cannot substitute for AID-dependent antibody gene diversification by class switch recombination. Regulatory differences between A3B and AID are also visible during cell cycle progression. Our studies suggest that the present-day A3B enzyme retained the nuclear import mechanism of an ancestral AID protein during the expansion of the APOBEC3 locus in primates. Our studies also highlight the likelihood that, after nuclear import, specialized mechanisms exist to guide these enzymes to their respective physiological substrates and prevent gratuitous chromosomal DNA damage.


Assuntos
Núcleo Celular/metabolismo , Citidina Desaminase/metabolismo , Transporte Ativo do Núcleo Celular , Motivos de Aminoácidos , Animais , Citidina Desaminase/química , Citidina Desaminase/genética , Evolução Molecular , Loci Gênicos , Células HeLa , Humanos , Antígenos de Histocompatibilidade Menor , Primatas/genética , Primatas/metabolismo
19.
RNA ; 11(3): 285-93, 2005 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-15661846

RESUMO

RNA-guided nucleotide modification complexes direct the post-transcriptional nucleotide modification of both archaeal and eukaryotic RNAs. We have previously demonstrated that efficient 2'-O-methylation activity guided by an in vitro reconstituted archaeal box C/D sRNP requires juxtaposed box C/D and C'/D' RNP complexes. In these experiments, we investigate the importance of spatially positioning the box C/D and C'/D' RNPs within the sRNP complex for nucleotide modification. Initial sequence analysis of 245 archaeal box C/D sRNAs from both Eukyarchaeota and Crenarchaeota kingdoms revealed highly conserved spacing between the box C/D and C'/D' RNA motifs. Distances between boxes C to D' and C' to D (D' and D spacers, respectively) exhibit highly constrained lengths of 12 nucleotides (nt). Methanocaldococcus jannaschii sR8 sRNA, a model box C/D sRNA with D and D' spacers of 12 nt, was mutated to alter the distance between the two RNA motifs. sRNAs with longer or shorter spacer regions could still form sRNPs by associating with box C/D core proteins, L7, Nop56/58, and fibrillarin, comparable to wild-type sR8. However, these reconstituted box C/D sRNP complexes were severely deficient in methylation activity. Alteration of the D and D' spacer lengths disrupted the guided methylation activity of both the box C/D and C'/D' RNP complexes. When only one spacer region was altered, methylation activity of the corresponding RNP was lost. Collectively, these results demonstrate the importance of box C/D and C'/D' RNP positioning for preservation of critical inter-RNP interactions required for efficient box C/D sRNP-guided nucleotide methylation.


Assuntos
Proteínas Arqueais/metabolismo , RNA Arqueal/metabolismo , Ribonucleoproteínas/metabolismo , Sequência de Bases , Primers do DNA , Metilação , Conformação de Ácido Nucleico , RNA Arqueal/química
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