title: Coordinated Action of Two Double-Stranded RNA Binding Motifs and an RGG Motif Enables Nuclear Factor 90 To Flexibly Target Different RNA Substrates.
abstract: The mechanisms of how RNA binding proteins (RBP) bind to and distinguish different RNA molecules are yet uncertain. Here, we performed a comprehensive analysis of the RNA binding properties of multidomain RBP nuclear factor 90 (NF90) by investigating specifically the functional activities of two double-stranded RNA binding motifs (dsRBM) and an RGG motif in the protein's unstructured C-terminus. By comparison of the RNA binding affinities of several NF90 variants and their modes of binding to a set of defined RNA molecules, the activities of the motifs turned out to be very different. While dsRBM1 contributes little to RNA binding, dsRBM2 is essential for effective binding of double-stranded RNA. The protein's immediate C-terminus, including the RGG motif, is indispensable for interactions of the protein with single-stranded RNA, and the RGG motif decisively contributes to NF90's overall RNA binding properties. Conformational studies, which compared wild-type NF90 with a variant that contains a pseudophosphorylated residue in the RGG motif, suggest that the NF90 C-terminus is involved in conformational changes in the protein after RNA binding, with the RGG motif acting as a central regulatory element. In summary, our data propose a concerted action of all RNA binding motifs within the frame of the full-length protein, which may be controlled by regulation of the activity of the RGG motif, e.g., by phosphorylation. This multidomain interplay enables the RBP NF90 to discriminate RNA features by dynamic and adaptable interactions.
authors.affiliation: Institute of Biochemistry and Biotechnology (NFI), Section of Microbial Biotechnology, and ‡Section of Protein Biochemistry, Martin Luther University Halle-Wittenberg , Kurt-Mothes-Strasse 3, D-06120 Halle/Saale, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Schmidt
authors.affiliation: Institute of Biochemistry and Biotechnology (NFI), Section of Microbial Biotechnology, and ‡Section of Protein Biochemistry, Martin Luther University Halle-Wittenberg , Kurt-Mothes-Strasse 3, D-06120 Halle/Saale, Germany.
authors.forename: Paul
authors.initials: P
authors.lastname: Knick
authors.affiliation: Institute of Biochemistry and Biotechnology (NFI), Section of Microbial Biotechnology, and ‡Section of Protein Biochemistry, Martin Luther University Halle-Wittenberg , Kurt-Mothes-Strasse 3, D-06120 Halle/Saale, Germany.
authors.forename: Hauke
authors.initials: H
authors.lastname: Lilie
authors.affiliation: Institute of Biochemistry and Biotechnology (NFI), Section of Microbial Biotechnology, and ‡Section of Protein Biochemistry, Martin Luther University Halle-Wittenberg , Kurt-Mothes-Strasse 3, D-06120 Halle/Saale, Germany.
authors.forename: Susann
authors.initials: S
authors.lastname: Friedrich
authors.affiliation: Institute of Biochemistry and Biotechnology (NFI), Section of Microbial Biotechnology, and ‡Section of Protein Biochemistry, Martin Luther University Halle-Wittenberg , Kurt-Mothes-Strasse 3, D-06120 Halle/Saale, Germany.
authors.forename: Ralph Peter
authors.initials: RP
authors.lastname: Golbik
authors.affiliation: Institute of Biochemistry and Biotechnology (NFI), Section of Microbial Biotechnology, and ‡Section of Protein Biochemistry, Martin Luther University Halle-Wittenberg , Kurt-Mothes-Strasse 3, D-06120 Halle/Saale, Germany.
authors.forename: Sven-Erik
authors.initials: SE
authors.lastname: Behrens
mesh_terms: D001665:Binding Sites / Q000502:physiology; D018390:Gene Targeting; D006801:Humans; D050779:Nuclear Factor 90 Proteins / Q000235:genetics / Q000378:metabolism*; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D059372:Nucleotide Motifs / Q000502:physiology*; D012330:RNA, Double-Stranded / Q000235:genetics / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D050779:Nuclear Factor 90 Proteins; D009687:Nuclear Proteins; C501499:RBMY1A1 protein, human; D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins