Tethered function assays: an adaptable approach to study RNA regulatory proteins.
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.
Coller
Jeff
J
Center for RNA Molecular Biology, Case Western Reserve University, Cleveland, Ohio, USA.
Wickens
Marv
M
D020413:3' Untranslated Regions; D036022:Capsid Proteins / Q000378:metabolism; D017930:Genes, Reporter / Q000502:physiology; D005821:Genetic Techniques; D035925:Iron-Regulatory Proteins / Q000378:metabolism; D017909:Levivirus; D012333:RNA, Messenger / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D017412:Ribonucleoprotein, U1 Small Nuclear / Q000378:metabolism; D054334:Viral Regulatory and Accessory Proteins / Q000378:metabolism
D020413:3' Untranslated Regions; D036022:Capsid Proteins; D035925:Iron-Regulatory Proteins; C059417:N protein, Bacteriophage lambda; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D017412:Ribonucleoprotein, U1 Small Nuclear; C075339:U1A protein; D054334:Viral Regulatory and Accessory Proteins