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Related Experiment Videos

Identification and characterization of proteins binding A + U-rich elements.

G M Wilson1, G Brewer

  • 1Department of Microbiology and Immunology, Wake Forest University School of Medicine, Medical Center Boulevard, Winston-Salem, North Carolina 27157, USA.

Methods (San Diego, Calif.)
|March 17, 1999
PubMed
Summary

This study details methods for identifying and characterizing AU-rich element (ARE)-binding proteins, specifically AUF1. These techniques enable the analysis of RNA-protein interactions and are applicable to other sequence-specific RNA-binding proteins.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Genetics

Background:

  • A+U-rich elements (AREs) are crucial cis-acting regulators of rapid mRNA turnover.
  • Several RNA-binding proteins interacting with AREs have been identified.
  • Understanding these interactions is key to deciphering gene expression regulation.

Purpose of the Study:

  • To present laboratory strategies and techniques for identifying and characterizing AUF1, a family of ARE-binding proteins.
  • To provide methods applicable to any protein with sequence-specific RNA binding activity.
  • To facilitate the study of RNA-protein interactions.

Main Methods:

  • Purification of native AUF1 from cultured cells.
  • Preparation of recombinant AUF1 proteins via bacterial expression system.

Related Experiment Videos

  • Gel mobility shift assays with synthetic RNA probes to monitor RNA binding activity.
  • Antibody supershifts for identifying proteins in RNA:protein complexes.
  • UV crosslinking for estimating molecular weights of RNA-binding proteins.

    • Main Results:

    • Demonstration of techniques for purifying and characterizing AUF1.
    • Validation of gel mobility shift assays for assessing RNA binding specificity and affinity.
    • Successful application of antibody supershifts and UV crosslinking for protein identification and molecular weight estimation.
    • Presentation of experimental results as examples for each technique.

    Conclusions:

    • The presented techniques are effective for the identification and characterization of AUF1.
    • These methods are versatile and can be optimized for studying other RNA-binding proteins.
    • The study provides a valuable toolkit for researchers investigating RNA-protein interactions and their role in gene regulation.