title: Recombinant hTRBP and hPACT Modulate hAgo2-Catalyzed siRNA-Mediated Target RNA Cleavage In Vitro.
abstract: The human TAR RNA-binding protein (hTRBP) and protein activator of protein kinase R (hPACT) are important players in RNA interference (RNAi). Together with hArgonaute2 (hAgo2) and hDicer they have been reported to form the RISC-loading complex (RLC). Among other functions, hTRBP was suggested to assist the loading of hAgo2 with small interfering RNAs (siRNAs) within the RLC. Although several studies have been conducted to evaluate the specific functions of hTRBP and hPACT in RNAi, exact mechanisms and modes of action are still unknown. Here, we present a biochemical study further evaluating the role of hTRBP and hPACT in hAgo2-loading. We found that both proteins enhance hAgo2-mediated RNA cleavage significantly; even a hAgo2 mutant impaired in siRNA binding shows full cleavage activity in the presence of hTRBP or hPACT. Pre-steady state binding studies reveal that the assembly of wildtype-hAgo2 (wt-hAgo2) and siRNAs remains largely unaffected, whereas the binding of mutant hAgo2-PAZ9 to siRNA is restored by adding either hTRBP or hPACT. We conclude that both proteins assist in positioning the siRNA within hAgo2 to ensure optimal binding and cleavage. Overall, our data indicate that hTRBP and hPACT are part of a regulative system of RNAi that is important for efficient target RNA cleavage.
authors.affiliation: Institute of Microbiology, Single Molecule Biochemistry Lab, University of Regensburg, Regensburg, 93053, Germany.|Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Lübeck, 23538, Germany.
authors.forename: Sarah
authors.initials: S
authors.lastname: Willkomm
authors.affiliation: Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Lübeck, 23538, Germany.
authors.forename: Andrea
authors.initials: A
authors.lastname: Deerberg
authors.affiliation: Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Lübeck, 23538, Germany.
authors.forename: Johannes
authors.initials: J
authors.lastname: Heidemann
authors.affiliation: Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Lübeck, 23538, Germany.
authors.forename: Friedemann
authors.initials: F
authors.lastname: Flügge
authors.affiliation: Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Lübeck, 23538, Germany.
authors.forename: Janica
authors.initials: J
authors.lastname: Meine
authors.affiliation: Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Lübeck, 23538, Germany.
authors.forename: Rui
authors.initials: R
authors.lastname: Hu
authors.affiliation: Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Lübeck, 23538, Germany.
authors.forename: Rosel
authors.initials: R
authors.lastname: Kretschmer-Kazemi Far
authors.affiliation: Institute of Molecular Medicine, Universitätsklinikum Schleswig-Holstein, University of Lübeck, Lübeck, 23538, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Restle
mesh_terms: D060565:Argonaute Proteins / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D011485:Protein Binding; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics; D016601:RNA-Binding Proteins / Q000378:metabolism*; D011994:Recombinant Proteins; D000099253:Trans-Activation Responsive RNA-Binding Protein
chemical_list: D060565:Argonaute Proteins; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; D011994:Recombinant Proteins; D000099253:Trans-Activation Responsive RNA-Binding Protein; C466422:AGO2 protein, human; C104647:PRKRA protein, human