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Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions
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Identification of RNA-Binding Proteins Associated to RNA Structural Elements.

Javier Fernandez-Chamorro1,2, Rosario Francisco-Velilla1, Azman Embarc-Buh1

  • 1Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.

Methods in Molecular Biology (Clifton, N.J.)
|June 4, 2021
PubMed
Summary

This study presents a novel pull-down assay to identify proteins that bind to specific RNA structural domains. The method helps uncover new factors involved in RNA and protein metabolism.

Keywords:
IRES elementsPull-downRNA structureRNA-binding proteinsStreptavidin aptamerTranslation controltRNA scaffold

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • Ribonucleoprotein complexes are crucial for cellular functions.
  • Internal ribosome entry site (IRES) elements regulate translation initiation through structural domains.

Purpose of the Study:

  • To describe a pull-down assay for identifying novel protein interactors of RNA structural domains.
  • To leverage RNA structure-dependent protein isolation for proteomic analysis.

Main Methods:

  • Utilizing streptavidin-aptamer tagged RNAs for pull-down assays.
  • Combining RNA structure-based protein isolation with proteomic analysis.
  • Employing tRNA-scaffold guided expression for factor identification.

Main Results:

  • Successful identification of novel protein interactors that recognize specific RNA structural domains.
  • The assay effectively isolates proteins based on their interaction with RNA structures.
  • Identification of factors involved in RNA and protein metabolism networks.

Conclusions:

  • The described pull-down assay is effective for discovering RNA-binding proteins.
  • This method aids in understanding the roles of RNA structural domains in cellular processes.
  • The approach facilitates the study of networks involved in RNA and protein metabolism.