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Before a cell can divide, it must accurately replicate all of its chromosomes, including the DNA and its associated histone and non-histone proteins.  This process begins at numerous origins of replication during the S phase of the cell cycle in each of a cell’s chromosomes simultaneously. Certain nucleotides can act as origins of replication, but these sequences are not well defined - especially in complex, multi-cellular, eukaryotic species. The length of DNA that spans an origin...
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A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues
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N6-methyladenosine modification and METTL3 modulate enterovirus 71 replication.

Haojie Hao1,2, Sujuan Hao1,2, Honghe Chen1

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N6-methyladenosine (m6A) RNA modification is crucial for enterovirus 71 (EV71) replication. Host m6A machinery interacts with viral proteins, impacting viral RNA processing and replication efficiency.

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Area of Science:

  • Virology
  • Molecular Biology
  • Epigenetics

Background:

  • N6-methyladenosine (m6A) is a prevalent internal RNA modification impacting RNA metabolism.
  • m6A's role in viral RNA has been recently explored for viruses like HIV, HCV, and Zika.
  • The functional significance of m6A in enterovirus 71 (EV71) infection remains largely uncharacterized.

Purpose of the Study:

  • To investigate the role and mechanism of m6A modification in EV71 replication.
  • To identify specific m6A sites within the EV71 genome and their functional impact.
  • To elucidate the interaction between host m6A regulators and viral components.

Main Methods:

  • Analysis of m6A modification in EV71-infected cells.
  • Knockdown experiments targeting m6A methyltransferases and demethylases.
  • Site-directed mutagenesis of identified m6A sites in the viral genome.
  • Co-immunoprecipitation assays to study protein-protein interactions.

Main Results:

  • EV71 RNA undergoes m6A modification during infection, affecting host m6A machinery localization.
  • Knockdown of m6A methyltransferase reduced EV71 replication, while demethylase knockdown enhanced it.
  • Mutating identified m6A sites in the EV71 genome decreased viral replication.
  • METTL3 was found to interact with viral 3D polymerase, promoting its sumoylation and ubiquitination, thereby boosting replication.

Conclusions:

  • m6A modification is a critical host factor that positively regulates EV71 replication.
  • Specific m6A sites within the EV71 genome are essential for efficient viral replication.
  • The host m6A writer METTL3 interacts with viral 3D polymerase to enhance its post-translational modification and promote viral replication.