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The human interferon (IFN)-induced MxA protein is a key antiviral host restriction factor exhibiting broad antiviral activity against many RNA viruses, including highly pathogenic avian influenza A viruses (IAV) of the H5N1 and H7N7 subtype. To date the mechanism for how MxA exerts its antiviral activity is unclear, however, additional cellular factors are believed to be essential for this activity. To identify MxA cofactors we performed a genome-wide siRNA-based screen in human airway epithelial cells (A549) constitutively expressing MxA using an H5N1 reporter virus. These data were complemented with a proteomic screen to identify MxA-interacting proteins. The combined data identified SMARCA2, the ATPase subunit of the BAF chromatin remodeling complex, as a crucial factor required for the antiviral activity of MxA against IAV. Intriguingly, our data demonstrate that although SMARCA2 is essential for expression of some IFN-stimulated genes (ISGs), and the establishment of an antiviral state, it is not required for expression of MxA, suggesting an indirect effect on MxA activity. Transcriptome analysis of SMARCA2-depleted A549-MxA cells identified a small set of SMARCA2-regulated factors required for activity of MxA, in particular IFITM2 and IGFBP3. These findings reveal that several virus-inducible factors work in concert to enable MxA restriction of IAV.
Assuntos
Virus da Influenza A Subtipo H5N1/crescimento & desenvolvimento , Vírus da Influenza A Subtipo H7N7/crescimento & desenvolvimento , Influenza Humana/virologia , Proteínas de Resistência a Myxovirus/metabolismo , Fatores de Transcrição/metabolismo , Células A549 , Antivirais/farmacologia , Perfilação da Expressão Gênica , Interações Hospedeiro-Patógeno , Humanos , Virus da Influenza A Subtipo H5N1/efeitos dos fármacos , Vírus da Influenza A Subtipo H7N7/efeitos dos fármacos , Influenza Humana/tratamento farmacológico , Influenza Humana/metabolismo , Interferons/farmacologia , Proteínas de Resistência a Myxovirus/genética , Proteoma/análise , Fatores de Transcrição/genética , Replicação ViralRESUMO
While low-throughput RNA bisulfite sequencing is the method of choice to assess the methylation status of specific cytosines in candidate RNAs, the combination of bisulfite treatment of RNA with today's high-throughput sequencing techniques opens the door to methylation studies at nucleotide resolution on a transcriptome-wide scale. Below we describe a protocol for the transcriptome-wide analysis of total or fractionated poly(A)RNA in cells and tissues. Although the nature of the bisulfite sequencing protocol makes it comparably easy to translate from a low to a high-throughput approach, several critical points require attention before starting such a project. We describe a step-by-step protocol for planning and performing the experiment and analyzing the data.
Assuntos
5-Metilcitosina/química , Sequenciamento de Nucleotídeos em Larga Escala , RNA/química , RNA/genética , Transcriptoma , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Biblioteca Gênica , Metilação , RNA/isolamento & purificação , RNA Mensageiro/química , RNA Mensageiro/genética , RNA Mensageiro/isolamento & purificaçãoRESUMO
BACKGROUND: Recent work has identified and mapped a range of posttranscriptional modifications in mRNA, including methylation of the N6 and N1 positions in adenine, pseudouridylation, and methylation of carbon 5 in cytosine (m5C). However, knowledge about the prevalence and transcriptome-wide distribution of m5C is still extremely limited; thus, studies in different cell types, tissues, and organisms are needed to gain insight into possible functions of this modification and implications for other regulatory processes. RESULTS: We have carried out an unbiased global analysis of m5C in total and nuclear poly(A) RNA of mouse embryonic stem cells and murine brain. We show that there are intriguing differences in these samples and cell compartments with respect to the degree of methylation, functional classification of methylated transcripts, and position bias within the transcript. Specifically, we observe a pronounced accumulation of m5C sites in the vicinity of the translational start codon, depletion in coding sequences, and mixed patterns of enrichment in the 3' UTR. Degree and pattern of methylation distinguish transcripts modified in both embryonic stem cells and brain from those methylated in either one of the samples. We also analyze potential correlations between m5C and micro RNA target sites, binding sites of RNA binding proteins, and N6-methyladenosine. CONCLUSION: Our study presents the first comprehensive picture of cytosine methylation in the epitranscriptome of pluripotent and differentiated stages in the mouse. These data provide an invaluable resource for future studies of function and biological significance of m5C in mRNA in mammals.
