Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences

Volker Boehm, Jennifer V. Gerbracht, Marie-Charlotte Marx and Niels H. Gehring ()
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Volker Boehm: Institute for Genetics, University of Cologne
Jennifer V. Gerbracht: Institute for Genetics, University of Cologne
Marie-Charlotte Marx: Institute for Genetics, University of Cologne
Niels H. Gehring: Institute for Genetics, University of Cologne

Nature Communications, 2016, vol. 7, issue 1, 1-15

Abstract: Abstract The turnover of messenger RNAs (mRNAs) is a key regulatory step of gene expression in eukaryotic cells. Due to the complexity of the mammalian degradation machinery, the contribution of decay factors to the directionality of mRNA decay is poorly understood. Here we characterize a molecular tool to interrogate mRNA turnover via the detection of XRN1-resistant decay fragments (xrFrag). Using nonsense-mediated mRNA decay (NMD) as a model pathway, we establish xrFrag analysis as a robust indicator of accelerated 5′–3′ mRNA decay. In tethering assays, monitoring xrFrag accumulation allows to distinguish decapping and endocleavage activities from deadenylation. Moreover, xrFrag analysis of mRNA degradation induced by miRNAs, AU-rich elements (AREs) as well as the 3′ UTRs of cytokine mRNAs reveals the contribution of 5′–3′ decay and endonucleolytic cleavage. Our work uncovers formerly unrecognized modes of mRNA turnover and establishes xrFrag as a powerful tool for RNA decay analyses.

Date: 2016
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DOI: 10.1038/ncomms13691

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