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1.
Genes Dev ; 35(13-14): 1005-1019, 2021 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-34168039

RESUMO

N6-methyladenosine (m6A) is an abundant internal RNA modification, influencing transcript fate and function in uninfected and virus-infected cells. Installation of m6A by the nuclear RNA methyltransferase METTL3 occurs cotranscriptionally; however, the genomes of some cytoplasmic RNA viruses are also m6A-modified. How the cellular m6A modification machinery impacts coronavirus replication, which occurs exclusively in the cytoplasm, is unknown. Here we show that replication of SARS-CoV-2, the agent responsible for the COVID-19 pandemic, and a seasonal human ß-coronavirus HCoV-OC43, can be suppressed by depletion of METTL3 or cytoplasmic m6A reader proteins YTHDF1 and YTHDF3 and by a highly specific small molecule METTL3 inhibitor. Reduction of infectious titer correlates with decreased synthesis of viral RNAs and the essential nucleocapsid (N) protein. Sites of m6A modification on genomic and subgenomic RNAs of both viruses were mapped by methylated RNA immunoprecipitation sequencing (meRIP-seq). Levels of host factors involved in m6A installation, removal, and recognition were unchanged by HCoV-OC43 infection; however, nuclear localization of METTL3 and cytoplasmic m6A readers YTHDF1 and YTHDF2 increased. This establishes that coronavirus RNAs are m6A-modified and host m6A pathway components control ß-coronavirus replication. Moreover, it illustrates the therapeutic potential of targeting the m6A pathway to restrict coronavirus reproduction.


Assuntos
Coronavirus Humano OC43/fisiologia , Processamento Pós-Transcricional do RNA/genética , SARS-CoV-2/fisiologia , Replicação Viral/genética , Adenosina/análogos & derivados , Adenosina/genética , Adenosina/metabolismo , Linhagem Celular , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/virologia , Regulação da Expressão Gênica/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Metiltransferases/antagonistas & inibidores , Metiltransferases/metabolismo , Proteínas do Nucleocapsídeo , RNA Viral/metabolismo , Proteínas de Ligação a RNA/metabolismo , Replicação Viral/efeitos dos fármacos
2.
Proc Natl Acad Sci U S A ; 117(7): 3711-3717, 2020 02 18.
Artigo em Inglês | MEDLINE | ID: mdl-32015105

RESUMO

Mosquito-borne helminth infections are responsible for a significant worldwide disease burden in both humans and animals. Accordingly, development of novel strategies to reduce disease transmission by targeting these pathogens in the vector are of paramount importance. We found that a strain of Aedes aegypti that is refractory to infection by Dirofilaria immitis, the agent of canine heartworm disease, mounts a stronger immune response during infection than does a susceptible strain. Moreover, activation of the Toll immune signaling pathway in the susceptible strain arrests larval development of the parasite, thereby decreasing the number of transmission-stage larvae. Notably, this strategy also blocks transmission-stage Brugia malayi, an agent of human lymphatic filariasis. Our data show that mosquito immunity can play a pivotal role in restricting filarial nematode development and suggest that genetically engineering mosquitoes with enhanced immunity will help reduce pathogen transmission.


Assuntos
Aedes/imunologia , Aedes/parasitologia , Dirofilaria immitis/crescimento & desenvolvimento , Mosquitos Vetores/imunologia , Mosquitos Vetores/parasitologia , Aedes/genética , Animais , Proteínas de Insetos/genética , Proteínas de Insetos/imunologia , Larva/crescimento & desenvolvimento , Mosquitos Vetores/genética
3.
Proc Natl Acad Sci U S A ; 116(45): 22583-22590, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31636182

RESUMO

Gene expression is rapidly remodeled by infection and inflammation in part via transcription factor NF-κB activation and regulated protein synthesis. While protein synthesis is largely controlled by mRNA translation initiation, whether cellular translation elongation factors are responsive to inflammation and infection remains poorly understood. Here, we reveal a surprising mechanism whereby NF-κB restricts phosphorylation of the critical translation elongation factor eEF2, which catalyzes the protein synthesis translocation step. Upon exposure to NF-κB-activating stimuli, including TNFα, human cytomegalovirus infection, or double-stranded DNA, eEF2 phosphorylation on Thr56, which slows elongation to limit protein synthesis, and the overall abundance of eEF2 kinase (eEF2K) are reduced. Significantly, this reflected a p65 NF-κB subunit-dependent reduction in eEF2K pre-mRNA, indicating that NF-κB activation represses eEF2K transcription to decrease eEF2K protein levels. Finally, we demonstrate that reducing eEF2K abundance regulates protein synthesis in response to a bacterial toxin that inactivates eEF2. This establishes that NF-κB activation by diverse physiological effectors controls eEF2 activity via a transcriptional repression mechanism that reduces eEF2K polypeptide abundance to preclude eEF2 phosphorylation, thereby stimulating translation elongation and protein synthesis. Moreover, it illustrates how nuclear transcription regulation shapes translation elongation factor activity and exposes how eEF2 is integrated into innate immune response networks orchestrated by NF-κB.


Assuntos
DNA/metabolismo , Quinase do Fator 2 de Elongação/genética , Inflamação/metabolismo , Biossíntese de Proteínas , Fator de Transcrição RelA/metabolismo , Motivos de Aminoácidos , DNA/genética , Quinase do Fator 2 de Elongação/química , Quinase do Fator 2 de Elongação/metabolismo , Humanos , Inflamação/genética , NF-kappa B/genética , NF-kappa B/metabolismo , Fator 2 de Elongação de Peptídeos/genética , Fator 2 de Elongação de Peptídeos/metabolismo , Fosforilação , Fator de Transcrição RelA/genética
4.
Genes Dev ; 32(23-24): 1472-1484, 2018 12 01.
Artigo em Inglês | MEDLINE | ID: mdl-30463905

RESUMO

Modification of mRNA by N 6-adenosine methylation (m6A) on internal bases influences gene expression in eukaryotes. How the dynamic genome-wide landscape of m6A-modified mRNAs impacts virus infection and host immune responses remains poorly understood. Here, we show that type I interferon (IFN) production triggered by dsDNA or human cytomegalovirus (HCMV) is controlled by the cellular m6A methyltrasferase subunit METTL14 and ALKBH5 demethylase. While METTL14 depletion reduced virus reproduction and stimulated dsDNA- or HCMV-induced IFNB1 mRNA accumulation, ALKBH5 depletion had the opposite effect. Depleting METTL14 increased both nascent IFNB1 mRNA production and stability in response to dsDNA. In contrast, ALKBH5 depletion reduced nascent IFNB1 mRNA production without detectably influencing IFN1B mRNA decay. Genome-wide transcriptome profiling following ALKBH5 depletion identified differentially expressed genes regulating antiviral immune responses, while METTL14 depletion altered pathways impacting metabolic reprogramming, stress responses, and aging. Finally, we determined that IFNB1 mRNA was m6A-modified within both the coding sequence and the 3' untranslated region (UTR). This establishes that the host m6A modification machinery controls IFNß production triggered by HCMV or dsDNA. Moreover, it demonstrates that responses to nonmicrobial dsDNA in uninfected cells, which shape host immunity and contribute to autoimmune disease, are regulated by enzymes controlling m6A epitranscriptomic changes.


Assuntos
DNA/imunologia , Regulação da Expressão Gênica/genética , Sistema Imunitário/enzimologia , Imunidade Inata/genética , Interferon beta/genética , Metiltransferases/metabolismo , Homólogo AlkB 5 da RNA Desmetilase/genética , Homólogo AlkB 5 da RNA Desmetilase/metabolismo , Animais , Linhagem Celular , Chlorocebus aethiops , Citomegalovirus/imunologia , Perfilação da Expressão Gênica , Humanos , Interferon beta/metabolismo , Estabilidade de RNA/genética , Células Vero , Replicação Viral/genética
5.
Health Promot Pract ; 18(5): 734-740, 2017 09.
Artigo em Inglês | MEDLINE | ID: mdl-28812927

RESUMO

INTRODUCTION: Cancer disparities continue to exist in the United States. Community health advisors (CHAs) can play a critical role in addressing cancer disparities. The American Cancer Society (ACS) implemented a 3-year pilot CHA program in the South based on an evidence-based program to increase breast cancer screening. STUDY DESIGN: Evaluation assessed the extent to which ACS successfully implemented the program. Quantitative data were tracked and reported by ACS staff, and qualitative data were collected through focus groups and interviews with volunteer participants. SETTING/PARTICIPANTS: The pilot was implemented in 28 communities in nine states. ACS staff recruited volunteer community network partners (CNPs) as local advisory groups, and volunteer CHAs to conduct outreach, education, and screening navigation. MEASURES: Outcome measures included number of individuals educated and screened, and number of communities reaching education and screening targets. Process measures included number of volunteers recruited, number of communities reaching recruitment targets, and implementation process, challenges, and successes. RESULTS: A total of 383 CHAs were recruited and recruitment goals were met in 68%; 31,439 individuals were educated, and 93% of communities reached education goals. In all, 5,056 individuals were screened, but screening goals were attained in only 36% of communities. CONCLUSION: This pilot demonstrates the ability of ACS to adapt and disseminate an evidence-based program to fit into its volunteer-based outreach model. ACS built community network partnerships, recruited a cadre of volunteers, and trained them to conduct education and screening navigation.


Assuntos
American Cancer Society/organização & administração , Agentes Comunitários de Saúde/organização & administração , Detecção Precoce de Câncer/estatística & dados numéricos , Promoção da Saúde/organização & administração , Saúde Pública , Região dos Apalaches , Feminino , Humanos , Masculino , Objetivos Organizacionais , Projetos Piloto , Pesquisa Qualitativa , Sudeste dos Estados Unidos , Estados Unidos , Voluntários
6.
Methods Mol Biol ; 1328: 21-8, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26324427

RESUMO

Drosophila melanogaster oogenesis has emerged as an excellent model system to study multiple aspects of eukaryotic cell biology. Ovarian tissue can easily be isolated and analyzed through microscopy or biochemical and molecular biology techniques. Here we describe the isolation of ovarian tissues, techniques to enrich for egg chambers at distinct developmental stages, preparation of protein and nucleic acid extracts, and preparation for microscopic analysis of fixed tissues.


Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Biologia Molecular/métodos , Oogênese , Ovário , Animais , Drosophila melanogaster/genética , Feminino , Oócitos/crescimento & desenvolvimento
7.
Dev Dyn ; 244(10): 1276-85, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26214278

RESUMO

BACKGROUND: In Drosophila, the dorsal-ventral (D-V) axis of the oocyte is dependent on Gurken (Grk) protein distribution. This is achieved through the cytoplasmic localization of grk mRNA and regulation of its translation. During mid-late stages of oogenesis, grk mRNA and protein are localized to the dorsal-anterior of the oocyte, while unlocalized grk transcripts are translationally silenced. As females carrying mutations in the gene encoding the CPEB protein Orb lay ventralized eggs due to insufficient Grk levels, it seemed likely that cytoplasmic polyadenylation of grk transcripts may play a role in their translational regulation. RESULTS: We have found that grk is polyadenylated throughout oogenesis, with poly(A) tails of approximately 30-50 A residues. Hyperadenylated grk transcripts, with poly(A) tails of 50-90 As, are detected in late stage egg chambers, but they fail to accumulate in oocytes deficient in Orb or the poly(A) polymerase Wispy (Wisp). wisp females also lay weakly ventralized eggs, demonstrating that they produce inadequate amounts of Grk. Finally, unlocalized grk transcripts are also not appropriately hyperadenylated. CONCLUSIONS: Localized cytoplasmic polyadenylation of grk mRNA by Wisp and Orb is necessary to achieve appropriate Grk protein accumulation in the D/A corner of the oocyte during mid to late oogenesis.


Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Polinucleotídeo Adenililtransferase/metabolismo , Proteínas de Ligação a RNA/metabolismo , Fator de Crescimento Transformador alfa/genética , Animais , Padronização Corporal , Drosophila melanogaster/genética , Feminino , Masculino , Oogênese , Poliadenilação , Proteínas de Ligação a RNA/genética
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