Tethered Function Assays: An Adaptable Approach to Study RNA Regulatory Proteins

Jeff Coller, Marv Wickens

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

Proteins and protein complexes that regulate mRNA metabolism must possess two activities. They bind the mRNA, and then elicit some function, that is, regulate mRNA splicing, transport, localization, translation, or stability. These two activities can often reside in different proteins in a complex, or in different regions of a single polypeptide. Much can be learned about the function of the protein or complex once it is stripped of the constraints imposed by RNA binding. With this in mind, we developed a "tethered function" assay, in which the mRNA regulatory protein is brought to the 3′ UTR of an mRNA reporter through a heterologous RNA-protein interaction. In this manner, the functional activity of the protein can be studied independent of its intrinsic ability to recognize and bind to RNA. This simple assay has proven useful in dissecting numerous proteins involved in posttranscriptional regulation. We discuss the basic assay, consider technical issues, and present case studies that exemplify the strengths and limitations of the approach.

Original languageEnglish (US)
Title of host publicationTranslation Initiation
Subtitle of host publicationExtract Systems and Molecular Genetics
PublisherAcademic Press Inc.
Pages299-321
Number of pages23
ISBN (Print)9780123741912
DOIs
StatePublished - 2007
Externally publishedYes

Publication series

NameMethods in Enzymology
Volume429
ISSN (Print)0076-6879

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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