Temporal and spatial characterization of nonsense-mediated mRNA decay

  1. Lynne E. Maquat2,3,6
  1. 1Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461, USA;
  2. 2Department of Biochemistry and Biophysics, School of Medicine and Dentistry, University of Rochester, Rochester, New York 14642, USA;
  3. 3Center for RNA Biology, University of Rochester, Rochester, New York 14642, USA;
  4. 4Gruss-Lipper Biophotonics Center, Albert Einstein College of Medicine, Bronx, New York 10461, USA
    • 5 Present address: Howard Hughes Medical Institute, Skirball Institute, New York University, Developmental Genetics Program, 540 First Avenue, 4th Floor, Lab 10, New York, New York, 10016.

    Abstract

    Nonsense-mediated mRNA decay (NMD) is a quality control mechanism responsible for “surveying” mRNAs during translation and degrading those that harbor a premature termination codon (PTC). Currently the intracellular spatial location of NMD and the kinetics of its decay step in mammalian cells are under debate. To address these issues, we used single-RNA fluorescent in situ hybridization (FISH) and measured the NMD of PTC-containing β-globin mRNA in intact single cells after the induction of β-globin gene transcription. This approach preserves temporal and spatial information of the NMD process, both of which would be lost in an ensemble study. We determined that decay of the majority of PTC-containing β-globin mRNA occurs soon after its export into the cytoplasm, with a half-life of <1 min; the remainder is degraded with a half-life of >12 h, similar to the half-life of normal PTC-free β-globin mRNA, indicating that it had evaded NMD. Importantly, NMD does not occur within the nucleoplasm, thus countering the long-debated idea of nuclear degradation of PTC-containing transcripts. We provide a spatial and temporal model for the biphasic decay of NMD targets.

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    Footnotes

    • 6 Corresponding authors

      E-mail robert.singer{at}einstein.yu.edu

      E-mail lynne_maquat{at}urmc.rochester.edu

    • Supplemental material is available for this article.

    • Article published online ahead of print. Article and publication date are online at http://www.genesdev.org/cgi/doi/10.1101/gad.209635.112.

    • Received November 6, 2012.
    • Accepted January 29, 2013.
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