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High-Throughput Micro-Characterization of RNA–Protein Interactions

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Part of the Methods in Molecular Biology book series (MIMB,volume 2025)

Abstract

Many cellular processes depend on and are regulated by nucleic acid–protein interactions. In particular, RNA-binding proteins (RBPs) are involved in transcription, translation, modulating RNA polymerase activity, and stabilizing protein–RNA complexes. Furthermore, RBPs participate in the development of pathologies such as cancer and viral infections, and their dysfunction leads to mutations and the aberrant expression of noncoding RNAs. Therefore, the study of RNA–protein interactions represents a central issue for biology and biomedicine. While many valuable insights have been obtained from electrophoretic mobility shift assays (EMSA) and immunoprecipitation (IP), these standard methods suffer from two main limitations: insufficient sensitivity to capture low concentration RBP–RNA complexes in vitro and identification of interactions in vivo. In recent years, high-throughput (HTP) platforms have emerged that combine methodological improvements over conventional techniques with more sensitive detection systems, thereby catalyzing the simultaneous probing and analysis of a vast amount of RBP–RNA interactions by cellular proteomics and interactomics approaches. In this chapter, we summarize a selection of state-of-the-art in vitro, in vivo, and computational HTP platforms for the discovery and characterization of RNA–protein interactions. We also reflect on the wealth of information obtained by the structural analysis of RBPs and their RNA-binding domains as a valuable resource for the rational design and implementation of new RNA-binding discovery platforms.

Key words

  • RNA
  • Protein
  • Interaction
  • RNA-binding domain
  • High-throughput assay
  • Micro-characterization

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Acknowledgments

We gratefully acknowledge the support received during the preparation of this chapter. MCV has received funding from the Spanish Ministerio de Economía y Competitividad (CTQ2015-66206-C2-2-R and SAF2015-72961-EXP) and the Regional Government of Madrid (S2017/BMD-3673). Abvance Biotech srl contributed with salaries (FJF). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Gómez, S., Fernández, F.J., Vega, M.C. (2019). High-Throughput Micro-Characterization of RNA–Protein Interactions. In: Vincentelli, R. (eds) High-Throughput Protein Production and Purification. Methods in Molecular Biology, vol 2025. Humana, New York, NY. https://doi.org/10.1007/978-1-4939-9624-7_24

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