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1.
2.
J Chem Theory Comput ; 16(1): 688-699, 2020 Jan 14.
Artigo em Inglês | MEDLINE | ID: mdl-31751512

RESUMO

Argonaute (Ago) protein plays a central role in silencing gene expression by binding a "guide" strand to the base-pair with a complementary mRNA and degrading the mRNA. The current understanding of how Ago-guide and Ago-guide-mRNA complexes assemble is based mainly on static crystal structures; the associated kinetic pathways remain unknown/unclear. By simulating the successive binding of guide/target strand to Thermus thermophilus Ago (TtAgo) and computing the respective free energy landscapes, we directly visualize how TtAgo silencing complexes form and function. We show that the guide binding rate depends on its initial loading position onto TtAgo. Subsequent target recognition beyond the scissile 10-11 nucleotides must overcome a substantial energy barrier for TtAgo's nucleotide-binding groove to expand widely. This work reveals novel roles for the core TtAgo domains and shows how the kinetic barriers that must be overcome for critical structural changes to occur lead to target repression/cleavage.


Assuntos
Proteínas Argonauta/metabolismo , Proteínas de Bactérias/metabolismo , RNA Mensageiro/metabolismo , Thermus thermophilus/metabolismo , Proteínas Argonauta/química , Proteínas de Bactérias/química , Inativação Gênica , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Estabilidade de RNA , RNA Bacteriano/química , RNA Bacteriano/metabolismo , RNA Mensageiro/química , Termodinâmica , Thermus thermophilus/química , Thermus thermophilus/genética
3.
J Clin Pathol ; 73(1): 14-16, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31434698

RESUMO

AIMS: Untranslated regions (UTRs) play an important role in post-transcriptional regulation of gene expression, including by modulating messenger RNA (mRNA) transport out of the nucleus, translation efficiency, subcellular localisation and stability. Any mutation in this region could alter the stability of mRNA and thereby affect protein synthesis. We analysed if a mutation located in the α complex protected region of the α1 globin gene could cause non-deletional α-thalassaemia by affecting post-transcriptional stability (mRNA stability). METHODS: A total of 14 patients without anaemia, normal or slight microcytosis and hypochromia (medium concentration haemoglobin [MCH] <27 pg) were studied. Haemoglobin subtypes were screened using capillary zone electrophoresis and ion-exchange high-performance liquid chromatography (VARIANT II ß-Thalassaemia Short Program). The most common α-globin mutations were identified by multiplex PCR (Alpha-Globin StripAssay kit) and the molecular characterisation by automatic sequencing of alpha globin genes. RESULTS: All of them shown a novel transversion mutation in nt 778 (C>A), which is located in the 3' UTR in the α complex protected region [HBA1: c.*+46C>A]. CONCLUSIONS: This mutation is in the αRNAmin binding site, so a single nucleotide substitution in this region can decrease mRNA stability by potentially compromising the binding of α-complex protein to αRNAmin, favouring the decay of α-globin mRNA via erythroid cell-enriched endoribonuclease cleavage. In this case, it is a non-deletional α-thalassaemia. However, in silico and empirical studies predicted that it could be a silent polymorphism. Functional studies should be carried out to confirm whether it is a pathological mutation or a silent polymorphism.


Assuntos
Regiões 3' não Traduzidas , Mutação , Polimorfismo Genético , Estabilidade de RNA , RNA Mensageiro/genética , alfa-Globinas/genética , Talassemia alfa/genética , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Análise Mutacional de DNA/métodos , Feminino , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase Multiplex , Fenótipo , RNA Mensageiro/metabolismo , Fatores de Risco , alfa-Globinas/metabolismo , Talassemia alfa/sangue , Talassemia alfa/diagnóstico
4.
Nat Genet ; 51(12): 1714-1722, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31784732

RESUMO

Core regulatory transcription factors (CR TFs) orchestrate the placement of super-enhancers (SEs) to activate transcription of cell-identity specifying gene networks, and are critical in promoting cancer. Here, we define the core regulatory circuitry of rhabdomyosarcoma and identify critical CR TF dependencies. These CR TFs build SEs that have the highest levels of histone acetylation, yet paradoxically the same SEs also harbor the greatest amounts of histone deacetylases. We find that hyperacetylation selectively halts CR TF transcription. To investigate the architectural determinants of this phenotype, we used absolute quantification of architecture (AQuA) HiChIP, which revealed erosion of native SE contacts, and aberrant spreading of contacts that involved histone acetylation. Hyperacetylation removes RNA polymerase II (RNA Pol II) from core regulatory genetic elements, and eliminates RNA Pol II but not BRD4 phase condensates. This study identifies an SE-specific requirement for balancing histone modification states to maintain SE architecture and CR TF transcription.


Assuntos
Histonas/metabolismo , Rabdomiossarcoma/genética , Fatores de Transcrição/genética , Acetilação , Benzamidas/farmacologia , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Elementos Facilitadores Genéticos , Proteína Forkhead Box O1/genética , Proteína Forkhead Box O1/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/genética , Humanos , Piridinas/farmacologia , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , Estabilidade de RNA , Fatores de Transcrição SOXE/genética , Análise de Célula Única
5.
Nat Commun ; 10(1): 4440, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31570709

RESUMO

The IGF2 mRNA-binding proteins (ZBP1/IMP1, IMP2, IMP3) are highly conserved post-transcriptional regulators of RNA stability, localization and translation. They play important roles in cell migration, neural development, metabolism and cancer cell survival. The knockout phenotypes of individual IMP proteins suggest that each family member regulates a unique pool of RNAs, yet evidence and an underlying mechanism for this is lacking. Here, we combine systematic evolution of ligands by exponential enrichment (SELEX) and NMR spectroscopy to demonstrate that the major RNA-binding domains of the two most distantly related IMPs (ZBP1 and IMP2) bind to different consensus sequences and regulate targets consistent with their knockout phenotypes and roles in disease. We find that the targeting specificity of each IMP is determined by few amino acids in their variable loops. As variable loops often differ amongst KH domain paralogs, we hypothesize that this is a general mechanism for evolving specificity and regulation of the transcriptome.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Sequência de Bases , Cristalografia por Raios X , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Ligantes , Espectroscopia de Ressonância Magnética , Camundongos , Modelos Moleculares , Mutação , Ligação Proteica , Domínios Proteicos , Estabilidade de RNA , RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/genética , Ribonucleoproteínas Nucleolares Pequenas , Técnica de Seleção de Aptâmeros , Transcriptoma
6.
J Microbiol ; 57(10): 910-917, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31571126

RESUMO

Studies have shown that many enzymes involved in glycolysis are upregulated in Escherichia coli endoribonuclease G (rng) null mutants. However, the molecular mechanisms underlying the RNase G-associated regulation of glycolysis have not been characterized. Here, we show that RNase G cleaves the 5' untranslated region of triosephosphate isomerase A (tpiA) mRNA, leading to destabilization of the mRNA in E. coli. Nucleotide substitutions within the RNase G cleavage site in the genome resulted in altered tpiA mRNA stability, indicating that RNase G activity influences tpiA mRNA abundance. In addition, we observed that tpiA expression was enhanced, whereas that of RNase G was decreased, in E. coli cells grown anaerobically. Our findings suggest that RNase G negatively regulates tpiA mRNA abundance in response to oxygen availability in E. coli.


Assuntos
Endorribonucleases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/enzimologia , RNA Mensageiro/genética , Triose-Fosfato Isomerase/genética , Endorribonucleases/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Estabilidade de RNA , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Bacteriano/metabolismo , RNA Mensageiro/química , RNA Mensageiro/metabolismo , Triose-Fosfato Isomerase/metabolismo
7.
Nat Commun ; 10(1): 4181, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519907

RESUMO

The stability and quality of metazoan mRNAs are under microRNA (miRNA)-mediated and nonsense-mediated control. Although UPF1, a core mediator of nonsense-mediated mRNA decay (NMD), mediates the decay of target mRNA in a 3'UTR-length-dependent manner, the detailed mechanism remains unclear. Here, we suggest that 3'UTR-length-dependent mRNA decay is not mediated by nonsense mRNAs but rather by miRNAs that downregulate target mRNAs via Ago-associated UPF1/SMG7. Global analyses of mRNAs in response to UPF1 RNA interference in miRNA-deficient cells reveal that 3'UTR-length-dependent mRNA decay by UPF1 requires canonical miRNA targeting. The destabilization of miRNA targets is accomplished by the combination of Ago2 and UPF1/SMG7, which may recruit the CCR4-NOT deadenylase complex. Indeed, loss of the SMG7-deadenylase complex interaction increases the levels of transcripts regulated by UPF1-SMG7. This UPF1/SMG7-dependent miRNA-mediated mRNA decay pathway may enable miRNA targeting to become more predictable and expand the miRNA-mRNA regulatory network.


Assuntos
Proteínas de Transporte/metabolismo , Biologia Computacional/métodos , MicroRNAs/metabolismo , RNA Helicases/metabolismo , Estabilidade de RNA/fisiologia , Transativadores/metabolismo , Regiões 3' não Traduzidas/genética , Animais , Western Blotting , Proteínas de Transporte/genética , Regulação da Expressão Gênica/genética , Regulação da Expressão Gênica/fisiologia , Células HeLa , Humanos , Camundongos , MicroRNAs/genética , RNA Helicases/genética , Interferência de RNA/fisiologia , Estabilidade de RNA/genética , Transativadores/genética
8.
Nat Commun ; 10(1): 4198, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519929

RESUMO

Maintenance of high-turnover tissues such as the epidermis requires a balance between stem cell proliferation and differentiation. The molecular mechanisms governing this process are an area of investigation. Here we show that HNRNPK, a multifunctional protein, is necessary to prevent premature differentiation and sustains the proliferative capacity of epidermal stem and progenitor cells. To prevent premature differentiation of progenitor cells, HNRNPK is necessary for DDX6 to bind a subset of mRNAs that code for transcription factors that promote differentiation. Upon binding, these mRNAs such as GRHL3, KLF4, and ZNF750 are degraded through the mRNA degradation pathway, which prevents premature differentiation. To sustain the proliferative capacity of the epidermis, HNRNPK is necessary for RNA Polymerase II binding to proliferation/self-renewal genes such as MYC, CYR61, FGFBP1, EGFR, and cyclins to promote their expression. Our study establishes a prominent role for HNRNPK in maintaining adult tissue self-renewal through both transcriptional and post-transcriptional mechanisms.


Assuntos
Diferenciação Celular/fisiologia , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , RNA Mensageiro/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Proliferação de Células/fisiologia , Células Cultivadas , Proteína Rica em Cisteína 61/genética , Proteína Rica em Cisteína 61/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epiderme/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Estabilidade de RNA/genética , Estabilidade de RNA/fisiologia , Células-Tronco/citologia , Células-Tronco/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
9.
Nat Commun ; 10(1): 4302, 2019 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-31541109

RESUMO

Type III CRISPR-Cas multisubunit complexes cleave ssRNA and ssDNA. These activities promote the generation of cyclic oligoadenylate (cOA), which activates associated CRISPR-Cas RNases from the Csm/Csx families, triggering a massive RNA decay to provide immunity from genetic invaders. Here we present the structure of Sulfolobus islandicus (Sis) Csx1-cOA4 complex revealing the allosteric activation of its RNase activity. SisCsx1 is a hexamer built by a trimer of dimers. Each dimer forms a cOA4 binding site and a ssRNA catalytic pocket. cOA4 undergoes a conformational change upon binding in the second messenger binding site activating ssRNA degradation in the catalytic pockets. Activation is transmitted in an allosteric manner through an intermediate HTH domain, which joins the cOA4 and catalytic sites. The RNase functions in a sequential cooperative fashion, hydrolyzing phosphodiester bonds in 5'-C-C-3'. The degradation of cOA4 by Ring nucleases deactivates SisCsx1, suggesting that this enzyme could be employed in biotechnological applications.


Assuntos
Nucleotídeos de Adenina/química , Proteínas Associadas a CRISPR/química , Sistemas CRISPR-Cas , Endorribonucleases/química , Oligorribonucleotídeos/química , Estabilidade de RNA , Proteínas de Ligação a RNA/química , Sulfolobus/química , Sítio Alostérico , Proteínas de Bactérias/química , Sítios de Ligação , Proteínas Associadas a CRISPR/genética , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Cristalografia por Raios X , Proteínas de Membrana/química , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Proteínas de Ligação a RNA/genética , Ribonucleases/metabolismo , Sistemas do Segundo Mensageiro , Sulfolobus/genética
10.
Nat Commun ; 10(1): 4424, 2019 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-31562313

RESUMO

Plant microRNAs (miRNAs) associate with ARGONAUTE1 (AGO1) to direct post-transcriptional gene silencing and regulate numerous biological processes. Although AGO1 predominantly binds miRNAs in vivo, it also associates with endogenous small interfering RNAs (siRNAs). It is unclear whether the miRNA/siRNA balance affects miRNA activities. Here we report that FIERY1 (FRY1), which is involved in 5'-3' RNA degradation, regulates miRNA abundance and function by suppressing the biogenesis of ribosomal RNA-derived siRNAs (risiRNAs). In mutants of FRY1 and the nuclear 5'-3' exonuclease genes XRN2 and XRN3, we find that a large number of 21-nt risiRNAs are generated through an endogenous siRNA biogenesis pathway. The production of risiRNAs correlates with pre-rRNA processing defects in these mutants. We also show that these risiRNAs are loaded into AGO1, causing reduced loading of miRNAs. This study reveals a previously unknown link between rRNA processing and miRNA accumulation.


Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Proteínas Argonauta/metabolismo , MicroRNAs/metabolismo , Monoéster Fosfórico Hidrolases/metabolismo , RNA Ribossômico/metabolismo , RNA Interferente Pequeno/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Exorribonucleases/genética , Genes de Plantas , Mutagênese , Proteínas Nucleares/genética , Monoéster Fosfórico Hidrolases/genética , Interferência de RNA , Estabilidade de RNA
11.
Genes Dev ; 33(21-22): 1575-1590, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31537626

RESUMO

PIWI proteins utilize small RNAs called piRNAs to silence transposable elements, thereby protecting germline integrity. In planarian flatworms, PIWI proteins are essential for regeneration, which requires adult stem cells termed neoblasts. Here, we characterize planarian piRNAs and examine the roles of PIWI proteins in neoblast biology. We find that the planarian PIWI proteins SMEDWI-2 and SMEDWI-3 cooperate to degrade active transposons via the ping-pong cycle. Unexpectedly, we discover that SMEDWI-3 plays an additional role in planarian mRNA surveillance. While SMEDWI-3 degrades numerous neoblast mRNAs in a homotypic ping-pong cycle, it is also guided to another subset of neoblast mRNAs by antisense piRNAs and binds these without degrading them. Mechanistically, the distinct activities of SMEDWI-3 are primarily dictated by the degree of complementarity between target mRNAs and antisense piRNAs. Thus, PIWI proteins enable planarians to repurpose piRNAs for potentially critical roles in neoblast mRNA turnover.


Assuntos
Células-Tronco Adultas/metabolismo , Proteínas de Helminto/metabolismo , Planárias/citologia , Planárias/metabolismo , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Animais , Pareamento de Bases , Elementos de DNA Transponíveis , Imunoprecipitação , Ligação Proteica , Estabilidade de RNA
12.
Mol Immunol ; 114: 524-534, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31518856

RESUMO

The chemokine CXCL4 has been implicated in several immune diseases. Exposure of monocyte-derived dendritic cells (moDCs) to CXCL4 potentiates the production of inflammatory cytokines in the presence of TLR3 or TLR7/8 agonists. Here we investigated the transcriptional and post-transcriptional events underlying the augmented inflammatory responses in CXCL4-moDCs. Our results indicate that CXCL4-moDCs display an increased expression and secretion of IL-12, IL-23, IL-6 and TNF upon TLR3 activation. Analysis of the cytokine transcripts for the presence of AU-rich elements (ARE), motifs necessary for ARE-mediated mRNA decay, revealed that all these cytokine transcripts are, at least in silico, possibly regulated at the level of mRNA stability. In vitro assays confirmed that mRNA stability of IL6 and TNF, but not IL12B and IL23A, is increased in CXCL4-moDCs. We next screened the expression of ARE-binding proteins (ARE-BPs) and found that TLR stimulation of CXCL4-moDCs induced tristetraprolin (TTP or ZFP36). Increased TTP mRNA expression was found to be a consequence of TTP phospho-mediated inactivation, which over time causes the protein to degrade its own mRNA. Concomitantly with TTP inactivation, we observed increased MAPK p38 signalling, upstream of TTP, in stimulated CXCL4-moDCs. P38 inhibition restored TTP activation and subsequently reduced the production of inflammatory cytokines. Finally, TTP knockdown in moDCs resulted in an increased production of IL6 and TNF after TLR stimulation. Overall, our study shows that the pro-inflammatory phenotype of CXCL4-moDCs relies in part on enhanced cytokine mRNA stability dictated by TTP inactivation.


Assuntos
Citocinas/metabolismo , Células Dendríticas/metabolismo , Monócitos/metabolismo , Fator Plaquetário 4/metabolismo , RNA Mensageiro/metabolismo , Humanos , Interleucina-12/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Estabilidade de RNA/fisiologia , Transdução de Sinais/fisiologia , Tristetraprolina/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
13.
Nucleic Acids Res ; 47(19): 10400-10413, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31501867

RESUMO

Chromosomally-encoded toxin-antitoxin complexes are ubiquitous in bacteria and regulate growth through the release of the toxin component typically in a stress-dependent manner. Type II ribosome-dependent toxins adopt a RelE-family RNase fold and inhibit translation by degrading mRNAs while bound to the ribosome. Here, we present biochemical and structural studies of the Escherichia coli YoeB toxin interacting with both a UAA stop and an AAU sense codon in pre- and post-mRNA cleavage states to provide insights into possible mRNA substrate selection. Both mRNAs undergo minimal changes during the cleavage event in contrast to type II ribosome-dependent RelE toxin. Further, the 16S rRNA decoding site nucleotides that monitor the mRNA in the aminoacyl(A) site adopt different orientations depending upon which toxin is present. Although YoeB is a RelE family member, it is the sole ribosome-dependent toxin that is dimeric. We show that engineered monomeric YoeB is active against mRNAs bound to both the small and large subunit. However, the stability of monomeric YoeB is reduced ∼20°C, consistent with potential YoeB activation during heat shock in E. coli as previously demonstrated. These data provide a molecular basis for the ability of YoeB to function in response to thermal stress.


Assuntos
Toxinas Bacterianas/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Estabilidade Proteica , Ribonucleases/química , Sequência de Aminoácidos/genética , Toxinas Bacterianas/genética , Códon/química , Códon/genética , Dimerização , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Resposta ao Choque Térmico/genética , Estabilidade de RNA/genética , RNA Mensageiro , RNA Ribossômico 16S/genética , Ribonucleases/genética , Ribossomos/química , Ribossomos/genética
15.
Nat Commun ; 10(1): 4056, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492834

RESUMO

The introduction of insertion-deletions (INDELs) by non-homologous end-joining (NHEJ) pathway underlies the mechanistic basis of CRISPR-Cas9-directed genome editing. Selective gene ablation using CRISPR-Cas9 is achieved by installation of a premature termination codon (PTC) from a frameshift-inducing INDEL that elicits nonsense-mediated decay (NMD) of the mutant mRNA. Here, by examining the mRNA and protein products of CRISPR targeted genes in a cell line panel with presumed gene knockouts, we detect the production of foreign mRNAs or proteins in ~50% of the cell lines. We demonstrate that these aberrant protein products stem from the introduction of INDELs that promote internal ribosomal entry, convert pseudo-mRNAs (alternatively spliced mRNAs with a PTC) into protein encoding molecules, or induce exon skipping by disruption of exon splicing enhancers (ESEs). Our results reveal challenges to manipulating gene expression outcomes using INDEL-based mutagenesis and strategies useful in mitigating their impact on intended genome-editing outcomes.


Assuntos
Edição de Genes/métodos , Mutagênese , RNA Mensageiro/genética , Sequência de Aminoácidos , Sequência de Bases , Sistemas CRISPR-Cas , Linhagem Celular , Linhagem Celular Tumoral , Códon sem Sentido/genética , Mutação da Fase de Leitura , Regulação Neoplásica da Expressão Gênica , Técnicas de Inativação de Genes , Células HeLa , Humanos , Mutação INDEL , Estabilidade de RNA , RNA Mensageiro/química
16.
Mol Cell ; 75(6): 1218-1228.e6, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31494033

RESUMO

Viral and endogenous double-stranded RNA (dsRNA) is a potent trigger for programmed RNA degradation by the 2-5A/RNase L complex in cells of all mammals. This 2-5A-mediated decay (2-5AMD) is a conserved stress response switching global protein synthesis from homeostasis to production of interferons (IFNs). To understand this mechanism, we examined 2-5AMD in human cells and found that it triggers polysome collapse characteristic of inhibited translation initiation. We determined that translation initiation complexes and ribosomes purified from translation-arrested cells remain functional. However, spike-in RNA sequencing (RNA-seq) revealed cell-wide decay of basal mRNAs accompanied by rapid accumulation of mRNAs encoding innate immune proteins. Our data attribute this 2-5AMD evasion to better stability of defense mRNAs and positive feedback in the IFN response amplified by RNase L-resistant molecules. We conclude that 2-5AMD and transcription act in concert to refill mammalian cells with defense mRNAs, thereby "prioritizing" the synthesis of innate immune proteins.


Assuntos
Endorribonucleases/metabolismo , Biossíntese de Proteínas , Estabilidade de RNA , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/metabolismo , Transcrição Genética , Células A549 , Endorribonucleases/genética , Humanos , Imunidade Inata , RNA de Cadeia Dupla/genética , RNA Mensageiro/genética
17.
Mol Cell ; 75(6): 1203-1217.e5, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31494035

RESUMO

In response to foreign and endogenous double-stranded RNA (dsRNA), protein kinase R (PKR) and ribonuclease L (RNase L) reprogram translation in mammalian cells. PKR inhibits translation initiation through eIF2α phosphorylation, which triggers stress granule (SG) formation and promotes translation of stress responsive mRNAs. The mechanisms of RNase L-driven translation repression, its contribution to SG assembly, and its regulation of dsRNA stress-induced mRNAs are unknown. We demonstrate that RNase L drives translational shut-off in response to dsRNA by promoting widespread turnover of mRNAs. This alters stress granule assembly and reprograms translation by allowing translation of mRNAs resistant to RNase L degradation, including numerous antiviral mRNAs such as interferon (IFN)-ß. Individual cells differentially activate dsRNA responses revealing variation that can affect cellular outcomes. This identifies bulk mRNA degradation and the resistance of antiviral mRNAs as the mechanism by which RNase L reprograms translation in response to dsRNA.


Assuntos
Reprogramação Celular , Endorribonucleases/metabolismo , Interferon beta/biossíntese , Biossíntese de Proteínas , RNA Mensageiro/metabolismo , eIF-2 Quinase/metabolismo , Células A549 , Endorribonucleases/genética , Células HEK293 , Humanos , Interferon beta/genética , Estabilidade de RNA , RNA de Cadeia Dupla/genética , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/genética , eIF-2 Quinase/genética
18.
Clin Lab ; 65(8)2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31414752

RESUMO

BACKGROUND: Blood or plasma samples from rural areas are often transported under suboptimal conditions to central laboratories. The negative influence of different storage temperatures during transportation as well as long transportation times on the stability of unprotected HIV RNA is well known. Therefore, the correct and reliable quantification of HIV RNA might be very difficult. A stabilization solution for the storage and transportation of plasma samples was developed which stabilizes RNA for seven days up to 45°C without viral load changes. METHODS: Blood samples from HIV positive individuals were collected into EDTA containing tubes. The isolated plasma samples in Germany were pipetted into pre-prepared RNA stabilization tubes and incubated for seven days at 45°C. HIV-1 RNA quantification was performed on a HIV-1 LCx m 2000 system from Abbott and a Qiagen/Artus HI Virus-1 RG RT-PCR Kit on a Rotor-Gene Q PCR machine. In addition, plasma samples were collected and tested using existing SOP for storage and transportation in Nigeria. Plasma samples were treated with and without stabilization solution and the AMPLICOR HIV-1 MONITORTM test was used to determine viral load. RESULTS: Seventy-four stabilized plasma samples were tested in Germany and results were compared to those tested unprotected within two hours. No significant changes of viral load were detected up to seven days and 45°C in case of stabilized samples. In contrast RNA of the same unprotected samples was no longer detectable after one day at 45°C. Additionally, 22 plasma samples were investigated on day zero and under field conditions in Nigeria without changes of the viral load after seven days under given temperature conditions. CONCLUSIONS: No cooling chain is necessary for the storage and/or transportation of plasma samples treated with the new RNA stabilization solution for up to seven days. The use of this solution to preserve plasma RNA will be very helpful in countries where the environmental temperature is higher than 30°C, thus addressing the problem of unreliable viral load results due to suboptimal storage or transportation conditions. Further, the costs of storage and transportation of samples for viral load quantification could be significantly reduced.


Assuntos
Infecções por HIV/sangue , HIV-1/genética , Estabilidade de RNA/genética , RNA Viral/sangue , Manejo de Espécimes/métodos , Carga Viral/genética , Liofilização , Alemanha , Infecções por HIV/virologia , HIV-1/fisiologia , Temperatura Alta , Humanos , RNA Viral/genética , Reprodutibilidade dos Testes , Manejo de Espécimes/instrumentação , Fatores de Tempo
19.
BMC Plant Biol ; 19(1): 349, 2019 Aug 09.
Artigo em Inglês | MEDLINE | ID: mdl-31399044

RESUMO

BACKGROUND: AFP is a negative regulator of ABA signaling that promotes ABI5 protein degradation and weakens regulation of ABA signaling by targeting upstream genes of ABI5, and TaABI5 gene was seed-specific, and accumulated during wheat grain maturation and dormancy acquisition, which played an important role in seed dormancy; TaAFP has a conserved domain with AFP, so TaAFP may also play an important role in seed dormancy in wheat. RESULTS: Two allelic variants of TaAFP were identified on chromosome 2BS in common wheat, and designated as TaAFP-B1a and TaAFP-B1b. Sequence analysis showed a 4-bp deletion in the 5'UTR region of TaAFP-B1b compared with TaAFP-B1a. Based on the 4-bp deletion, a co-dominant functional marker of TaAFP-B was developed and designated as AFPB. The genotype generating a 203-bp fragment (TaAFP-B1b) was more resistant to pre-harvest sprouting than the genotype producing a 207-bp fragment (TaAFP-B1a) in a test of 91 white-grained Chinese wheat cultivars and advanced lines. The average germination index(GI) values of TaAFP-B1a and that of TaAFP-B1b were 45.18 and 30.72%, respectively, indicating a significant difference (P < 0.001). Moreover, the 4-bp deletion located in the 5'UTR not only affected the transcription level of TaAFP-B but also affected the mRNA decay, reduced the translation level of GUS and tdTomatoER and GUS activity in wheat leaves of transient expression. The transcript expression and the mRNA half-life value of TaAFP-B1a in developing seeds and mature seeds were much higher than those of TaAFP-B1b. CONCLUSION: We identified a 4-bp InDel in the 5'UTR of TaAFP-B, which affected the mRNA transcription level, mRNA decay, translation levels of GUS and tdTomatoER, GUS activity, and was significantly associated with seed dormancy in common wheat. A functional marker was developed and validated based on this InDel.


Assuntos
Dormência de Plantas/genética , Proteínas de Plantas/genética , Triticum/genética , Regiões 5' não Traduzidas/genética , Ácido Abscísico/metabolismo , Regulação da Expressão Gênica de Plantas , Desenvolvimento Vegetal/genética , Biossíntese de Proteínas , Estabilidade de RNA , RNA Mensageiro/metabolismo , Deleção de Sequência , Transdução de Sinais/genética , Triticum/crescimento & desenvolvimento
20.
Mol Cell ; 75(6): 1188-1202.e11, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31399345

RESUMO

The maternal-to-zygotic transition (MZT) is a conserved and fundamental process during which the maternal environment is converted to an environment of embryonic-driven development through dramatic reprogramming. However, how maternally supplied transcripts are dynamically regulated during MZT remains largely unknown. Herein, through genome-wide profiling of RNA 5-methylcytosine (m5C) modification in zebrafish early embryos, we found that m5C-modified maternal mRNAs display higher stability than non-m5C-modified mRNAs during MZT. We discovered that Y-box binding protein 1 (Ybx1) preferentially recognizes m5C-modified mRNAs through π-π interactions with a key residue, Trp45, in Ybx1's cold shock domain (CSD), which plays essential roles in maternal mRNA stability and early embryogenesis of zebrafish. Together with the mRNA stabilizer Pabpc1a, Ybx1 promotes the stability of its target mRNAs in an m5C-dependent manner. Our study demonstrates an unexpected mechanism of RNA m5C-regulated maternal mRNA stabilization during zebrafish MZT, highlighting the critical role of m5C mRNA modification in early development.


Assuntos
5-Metilcitosina/metabolismo , Embrião não Mamífero/embriologia , Desenvolvimento Embrionário/fisiologia , Estabilidade de RNA/fisiologia , RNA Mensageiro Estocado/metabolismo , Peixe-Zebra/embriologia , Animais , Células HeLa , Humanos , Camundongos , RNA Mensageiro Estocado/genética , Peixe-Zebra/genética
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