TY - JOUR
T1 - Tethered function assays using 3′ untranslated regions
AU - Coller, Jeffery
AU - Wickens, Marvin
N1 - Funding Information:
We thank many individuals who have contributed thoughts and ideas to this review, most notably, Dr. Scott Ballantyne, Dr. Kris Dickson, Dr. Stan Fields, Dr. Niki Gray, Dr. Matthias Hentze, Dr. Allan Jacobson, Dr. Roy Long, Dr. Jens Lykke-Anderson, Dr. Roy Parker, Dr. Stu Peltz, Dr. Daniel Seay, Dr. Rob Singer, Dr. Nancy Standart, and Dr. Joan Steitz. We are particularly grateful to Carol Pfeffer for help preparing the manuscript, and to the University of Wisconsin Biochemistry Media Laboratory for the figures. Some of the work referred to in this review was supported by a grant to Dr. Marvin Wickens from NIH (GM31892). Dr. Coller is now supported by the HHMI.
PY - 2002
Y1 - 2002
N2 - Proteins that regulate mRNA metabolism are often bipartite: an RNA binding activity confers substrate specificity, and a "functional" domain elicits the biological outcome. In some cases, these two activities reside on different polypeptides that form a complex on the mRNA. Regardless, experimental separation of RNA binding from function facilitates analysis of both properties, liberating each from the constraints of the other. "Tethered function" assays bring a protein to a reporter RNA through a designed RNA-protein interaction. The function of the tethered protein-whether that be stability, translation, localization, or transport, or otherwise - is then assessed. We refer to this approach as a "tethered function" assay, since it can be examined. The approach does not require knowledge of the natural RNA binding sites, or of the composition of the naturally occurring protein complexes. It can be useful in dissecting how proteins that act on RNAs work, and in identifying active components of multiprotein complexes. RNA-binding proteins previously have been linked to foreign RNA-binding specificities, for a wide variety of purposes. We emphasize here the particular value of tethering to the 3′ untranslated region of eukaryotic mRNAs, and the opportunities it presents for the analysis of how those mRNAs are regulated. We discuss experimental design, as well as published and potential applications.
AB - Proteins that regulate mRNA metabolism are often bipartite: an RNA binding activity confers substrate specificity, and a "functional" domain elicits the biological outcome. In some cases, these two activities reside on different polypeptides that form a complex on the mRNA. Regardless, experimental separation of RNA binding from function facilitates analysis of both properties, liberating each from the constraints of the other. "Tethered function" assays bring a protein to a reporter RNA through a designed RNA-protein interaction. The function of the tethered protein-whether that be stability, translation, localization, or transport, or otherwise - is then assessed. We refer to this approach as a "tethered function" assay, since it can be examined. The approach does not require knowledge of the natural RNA binding sites, or of the composition of the naturally occurring protein complexes. It can be useful in dissecting how proteins that act on RNAs work, and in identifying active components of multiprotein complexes. RNA-binding proteins previously have been linked to foreign RNA-binding specificities, for a wide variety of purposes. We emphasize here the particular value of tethering to the 3′ untranslated region of eukaryotic mRNAs, and the opportunities it presents for the analysis of how those mRNAs are regulated. We discuss experimental design, as well as published and potential applications.
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U2 - 10.1016/S1046-2023(02)00016-6
DO - 10.1016/S1046-2023(02)00016-6
M3 - Article
C2 - 12054890
AN - SCOPUS:0036352777
SN - 1046-2023
VL - 26
SP - 142
EP - 150
JO - ImmunoMethods
JF - ImmunoMethods
IS - 2
ER -