TY - CHAP
T1 - Tethered Function Assays
T2 - An Adaptable Approach to Study RNA Regulatory Proteins
AU - Coller, Jeff
AU - Wickens, Marv
N1 - Funding Information:
We thank many individuals who have contributed their thoughts and ideas to this review, most notably, Drs. Jens Lykke‐Anderson, Scott Ballantyne, Kristian Baker, Kris Dickson, Niki Gray, Stan Fields, Mattias Hentze, Allan Jacobson, Roy Parker, Stu Peltz, Daniel Seay, Rob Singer, Nancy Standart, and Joan Steiz. We also thank Drs. Wenqian Hu and Thomas J. Sweet for critical reading of the manuscript. Work in the Wickens laboratory is supported by grants from the National Institutes of Health (NIH). Dr. Coller is supported by a grant from the American Cancer Society and the NIH.
PY - 2007
Y1 - 2007
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=38449094651&partnerID=8YFLogxK
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U2 - 10.1016/S0076-6879(07)29014-7
DO - 10.1016/S0076-6879(07)29014-7
M3 - Chapter
C2 - 17913629
AN - SCOPUS:38449094651
SN - 9780123741912
T3 - Methods in Enzymology
SP - 299
EP - 321
BT - Translation Initiation
PB - Academic Press Inc.
ER -