RNA degradation ensures appropriate levels of mRNA transcripts within cells and eliminates aberrant RNAs. Detailed studies of RNA degradation dynamics have been heretofore infeasible because of the inherent instability of degradation intermediates due to the high processivity of the enzymes involved. To visualize decay intermediates and to characterize the spatiotemporal dynamics of mRNA decay, we have developed a set of methods that apply XRN1-resistant RNA sequences (xrRNAs) to protect mRNA transcripts from 5′–3′ exonucleolytic digestion. To our knowledge, this approach is the only method that can detect the directionality of mRNA degradation and that allows tracking of degradation products in unperturbed cells. Here, we provide detailed procedures for xrRNA reporter design, transfection and cell line generation. We explain how to extract xrRNA reporter mRNAs from mammalian cells, as well as their detection and quantification using northern blotting and quantitative PCR. The procedure further focuses on how to detect and quantify intact reporter mRNAs and XRN1-resistant degradation intermediates using single-molecule fluorescence microscopy. It provides detailed instructions for sample preparation and image acquisition using fixed, as well as living, cells. The procedure puts special emphasis on detailed descriptions of high-throughput image analysis pipelines, which are provided along with the article and were designed to perform spot co-localization, detection efficiency normalization and the quality control steps necessary for interpretation of results. The aim of the analysis software published here is to enable nonexpert readers to detect and quantify RNA decay intermediates within 4–6 d after reporter mRNA expression.
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Example datasets and analysis software are available from the Chao lab website (https://data.fmi.ch/PublicationSupplementRepo/?group=gchao).
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This work was funded by the Novartis Research Foundation (J.A.C.), the Institutional Strategy of the University of Cologne within the German Excellence Initiative (V.B.), a Swiss National Science Foundation (SNF) grant (31003A_156477; J.A.C.), a grant from the Deutsche Forschungsgemeinschaft (GE 2014/4-1; N.H.G), the SNF-NCCR RNA & Disease (J.A.C.) and an SNF Marie Heim-Vögtlin fellowship (F.V). N.H.G. acknowledges support from a Heisenberg fellowship (GE 2014/5-1 and GE 2014/7-1). We thank K. Schönig (CIMH) for the parental HeLa 11ht cell line, E. Dobrikova and M. Gromeier for establishing and M. Hentze for sharing the HeLa Flp-In T-REx cell line, L. Lavis (Janelia Farm) for providing Halo dyes, and L. Gelman and S. Bourke (FMI) for microscopy support, as well as H. Kohler (FMI) for cell sorting.
The authors declare no competing interests.
Journal peer review information: Nature Protocols thanks Marvin Tanenbaum and other anonymous reviewer(s) for their contribution to the peer review of this work.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Key references using this protocol
Boehm, V., Gerbracht, J. V., Marx, M.-C. & Gehring, N. H. Nat. Commun. 7, 13691 (2016): https://www.nature.com/articles/ncomms13691
Gerbracht, J. V., Boehm, V. & Gehring, N. H. Sci. Rep. 7, 10616 (2017): https://www.nature.com/articles/s41598-017-10847-4
Horvathova, I. et al. Mol. Cell 68, 615–625.e9 (2017): https://doi.org/10.1016/j.molcel.2017.09.030