title: Interrogating the degradation pathways of unstable mRNAs with XRN1-resistant sequences.
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.
authors.affiliation: Institute for Genetics, Department of Biology, University of Cologne, Zuelpicher Straße 47a, 50674 Cologne, Germany.
authors.forename: Volker
authors.identifier: 0000-0001-7588-9842
authors.initials: V
authors.lastname: Boehm
authors.affiliation: Institute for Genetics, Department of Biology, University of Cologne, Zuelpicher Straße 47a, 50674 Cologne, Germany.
authors.forename: Jennifer V
authors.initials: JV
authors.lastname: Gerbracht
authors.affiliation: Institute for Genetics, Department of Biology, University of Cologne, Zuelpicher Straße 47a, 50674 Cologne, Germany.
authors.forename: Marie-Charlotte
authors.initials: MC
authors.lastname: Marx
authors.affiliation: Institute for Genetics, Department of Biology, University of Cologne, Zuelpicher Straße 47a, 50674 Cologne, Germany.
authors.forename: Niels H
authors.identifier: 0000-0001-7792-1164
authors.initials: NH
authors.lastname: Gehring
mesh_terms: D020413:3' Untranslated Regions / Q000235:genetics; D063307:AU Rich Elements / Q000235:genetics; D001483:Base Sequence; D005095:Exoribonucleases / Q000235:genetics* / Q000378:metabolism; D015972:Gene Expression Regulation, Neoplastic; D006367:HeLa Cells; D006801:Humans; D015850:Interleukin-6 / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000235:genetics; D008869:Microtubule-Associated Proteins / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D059365:Nonsense Mediated mRNA Decay; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics* / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism
chemical_list: D020413:3' Untranslated Regions; D015850:Interleukin-6; D035683:MicroRNAs; D008869:Microtubule-Associated Proteins; D012333:RNA, Messenger; D014409:Tumor Necrosis Factor-alpha; D005095:Exoribonucleases; C470512:XRN1 protein, human