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      N6 -Methyladenosine in Flaviviridae Viral RNA Genomes Regulates Infection

      Cell Host & Microbe
      Elsevier BV

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          The lipid droplet is an important organelle for hepatitis C virus production.

          The lipid droplet (LD) is an organelle that is used for the storage of neutral lipids. It dynamically moves through the cytoplasm, interacting with other organelles, including the endoplasmic reticulum (ER). These interactions are thought to facilitate the transport of lipids and proteins to other organelles. The hepatitis C virus (HCV) is a causative agent of chronic liver diseases. HCV capsid protein (Core) associates with the LD, envelope proteins E1 and E2 reside in the ER lumen, and the viral replicase is assumed to localize on ER-derived membranes. How and where HCV particles are assembled, however, is poorly understood. Here, we show that the LD is involved in the production of infectious virus particles. We demonstrate that Core recruits nonstructural (NS) proteins and replication complexes to LD-associated membranes, and that this recruitment is critical for producing infectious viruses. Furthermore, virus particles were observed in close proximity to LDs, indicating that some steps of virus assembly take place around LDs. This study reveals a novel function of LDs in the assembly of infectious HCV and provides a new perspective on how viruses usurp cellular functions.
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            Transcriptome-wide mapping of N(6)-methyladenosine by m(6)A-seq based on immunocapturing and massively parallel sequencing.

            N(6)-methyladenosine-sequencing (m(6)A-seq) is an immunocapturing approach for the unbiased transcriptome-wide localization of m(6)A in high resolution. To our knowledge, this is the first protocol to allow a global view of this ubiquitous RNA modification, and it is based on antibody-mediated enrichment of methylated RNA fragments followed by massively parallel sequencing. Building on principles of chromatin immunoprecipitation-sequencing (ChIP-seq) and methylated DNA immunoprecipitation (MeDIP), read densities of immunoprecipitated RNA relative to untreated input control are used to identify methylated sites. A consensus motif is deduced, and its distance to the point of maximal enrichment is assessed; these measures further corroborate the success of the protocol. Identified locations are intersected in turn with gene architecture to draw conclusions regarding the distribution of m(6)A between and within gene transcripts. When applied to human and mouse transcriptomes, m(6)A-seq generated comprehensive methylation profiles revealing, for the first time, tenets governing the nonrandom distribution of m(6)A. The protocol can be completed within ~9 d for four different sample pairs (each consists of an immunoprecipitation and corresponding input).
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              m(6)A RNA methylation is regulated by microRNAs and promotes reprogramming to pluripotency.

              N(6)-methyladenosine (m(6)A) has been recently identified as a conserved epitranscriptomic modification of eukaryotic mRNAs, but its features, regulatory mechanisms, and functions in cell reprogramming are largely unknown. Here, we report m(6)A modification profiles in the mRNA transcriptomes of four cell types with different degrees of pluripotency. Comparative analysis reveals several features of m(6)A, especially gene- and cell-type-specific m(6)A mRNA modifications. We also show that microRNAs (miRNAs) regulate m(6)A modification via a sequence pairing mechanism. Manipulation of miRNA expression or sequences alters m(6)A modification levels through modulating the binding of METTL3 methyltransferase to mRNAs containing miRNA targeting sites. Increased m(6)A abundance promotes the reprogramming of mouse embryonic fibroblasts (MEFs) to pluripotent stem cells; conversely, reduced m(6)A levels impede reprogramming. Our results therefore uncover a role for miRNAs in regulating m(6)A formation of mRNAs and provide a foundation for future functional studies of m(6)A modification in cell reprogramming.
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                Author and article information

                Journal
                10.1016/j.chom.2016.09.015
                http://creativecommons.org/licenses/by/4.0/

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