Abstract
RNA-binding proteins and mitochondrial ribosomes have been found to be linchpins of mitochondrial gene expression in health and disease. The expanding repertoire of proteins that bind and regulate the mitochondrial transcriptome has necessitated the development of new tools and methods to examine their molecular functions. Next-generation sequencing technologies have advanced the RNA biology field through application of high-throughput methods to study RNA-protein interactions. Here we describe a digital RNase footprinting method to analyze protein and ribosome interactions with mitochondrially encoded transcripts that provides insight into their mechanisms and minimal binding sites. We provide details on RNase digestion and next-generation sequencing, along with computational analyses and visualization of the binding targets within the mitochondrial transcriptome.
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Acknowledgments
This project was supported by fellowships and project grants from the NHMRC, ARC, and CCWA (to OR and AF). DR was supported by a UWA Postgraduate Scholarship.
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© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
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Rudler, D.L., Siira, S.J., Rackham, O., Filipovska, A. (2023). Digital RNase Footprinting of RNA-Protein Complexes and Ribosomes in Mitochondria. In: Barrientos, A., Fontanesi, F. (eds) The Mitoribosome. Methods in Molecular Biology, vol 2661. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3171-3_18
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DOI: https://doi.org/10.1007/978-1-0716-3171-3_18
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