Abstract
Recent progress in the technology of transcriptome-wide high-throughput sequencing has revealed that nonsense-mediated mRNA decay (NMD) targets ~10% of physiologic transcripts for the purpose of tuning gene expression in response to various environmental conditions. Regardless of the eukaryote studied, NMD requires the ATP-dependent RNA helicase upframeshift 1 (UPF1). It was initially thought that cellular NMD targets could be defined by their binding to steady-state UPF1, which is largely hypophosphorylated. However, the propensity for steady-state UPF1 to bind RNA nonspecifically, coupled with regulated phosphorylation of UPF1 on an NMD target serving as the trigger for NMD, made it clear that it is phosphorylated UPF1 (p-UPF1), rather than steady-state UPF1, that can be used to distinguish cellular NMD targets from cellular RNAs that are not. Here, we describe the immunoprecipitation of p-UPF1 followed by RNA sequencing (p-UPF1 RIP−seq) as a transcriptome-wide approach to define physiologic NMD targets.
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Acknowledgments
We thank Keita Miyoshi for comments on the manuscript. Work on NMD in the Maquat lab is supported by NIH R01 GM059614 to L.E.M. T.K. was partially supported by a postdoctoral fellowship from FRAXA Research Foundation.
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Kurosaki, T., Hoque, M., Maquat, L.E. (2018). Identifying Cellular Nonsense-Mediated mRNA Decay (NMD) Targets: Immunoprecipitation of Phosphorylated UPF1 Followed by RNA Sequencing (p-UPF1 RIP−Seq). In: Lamandé, S. (eds) mRNA Decay. Methods in Molecular Biology, vol 1720. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7540-2_13
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DOI: https://doi.org/10.1007/978-1-4939-7540-2_13
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