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Global Identification of Human Exosome Substrates Using RNA Interference and RNA Sequencing

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Part of the Methods in Molecular Biology book series (MIMB, volume 2062)

Abstract

The RNA exosome is involved in RNA processing and quality control. In humans, it consists of an enzymatically inactive nine-subunit core, with ribonucleolytic activity contributed by one or two additional components. Moreover, several protein cofactors interact with the exosome to enable and specify its recruitment to a wide range of substrates. A common strategy to identify these substrates has been to deplete an exosome subunit or a cofactor and subsequently interrogate which transcripts become stabilized. Here, we describe an experimental pipeline including siRNA-mediated depletion of the RNA exosome or its cofactors in HeLa cells, confirmation of the knockdown efficiencies, and the manual or high-throughput identification of exosome targets.

Key words

RNA exosome RNAi RNA-seq Exosome cofactors Western blotting analysis RT-qPCR analysis 
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Notes

Acknowledgments

We thank Manfred Schmid for critical comments on the bioinformatics section of the manuscript. This work was supported by the Lundbeck and Novo Nordisk Foundations and the ERC (grant 339953).

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Authors and Affiliations

  1. 1.Department of Molecular Biology and GeneticsAarhus UniversityAarhusDenmark

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