Biochemical isolation of Argonaute protein complexes by Ago-APP.
During microRNA (miRNA)-guided gene silencing, Argonaute (Ago) proteins interact with a member of the TNRC6/GW protein family. Here we used a short GW protein-derived peptide fused to GST and demonstrate that it binds to Ago proteins with high affinity. This allows for the simultaneous isolation of all Ago protein complexes expressed in diverse species to identify associated proteins, small RNAs, or target mRNAs. We refer to our method as "Ago protein Affinity Purification by Peptides" (Ago-APP). Furthermore, expression of this peptide competes for endogenous TNRC6 proteins, leading to global inhibition of miRNA function in mammalian cells.
Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany;
Judith
J
Hauptmann
Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany;
Daniel
D
Schraivogel
Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany;
Astrid
A
Bruckmann
National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892;
Sudhir
S
Manickavel
Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany;
Leonhard
L
Jakob
Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany;
Norbert
N
Eichner
Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany;
Janina
J
Pfaff
Cell Biology and Plant Biochemistry, Biochemistry Center Regensburg, University of Regensburg, 93053 Regensburg, Germany;
Marc
M
Urban
Cell Biology and Plant Biochemistry, Biochemistry Center Regensburg, University of Regensburg, 93053 Regensburg, Germany;
Stefanie
S
Sprunck
National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892;
Markus
M
Hafner
Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, The Rockefeller University, New York, NY 10065.
Thomas
T
Tuschl
Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany;
Rainer
R
Deutzmann
Biochemistry Center Regensburg, Laboratory for RNA Biology, University of Regensburg, 93053 Regensburg, Germany; gunter.meister@vkl.uni-regensburg.de.
Gunter
G
Meister
D000595:Amino Acid Sequence; D000818:Animals; D060565:Argonaute Proteins / Q000302:isolation & purification*; D002457:Cell Extracts; D011232:Chemical Precipitation; D002846:Chromatography, Affinity / Q000379:methods*; D004331:Drosophila melanogaster; D020868:Gene Silencing; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D035683:MicroRNAs / Q000378:metabolism; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000302:isolation & purification*; D010455:Peptides / Q000737:chemistry / Q000302:isolation & purification*
D060565:Argonaute Proteins; D002457:Cell Extracts; D035683:MicroRNAs; D046912:Multiprotein Complexes; D010455:Peptides
Argonaute; GW proteins; RNAi; microRNAs; small RNAs