Comparative mutational analysis of the double-stranded RNA binding domains of Xenopus laevis RNA-binding protein A.
Xenopus laevis RNA-binding protein A is a ubiquitously expressed, double-stranded RNA-binding protein that is associated with the majority of cellular RNAs, ribosomal RNAs, and hnRNAs. X. laevis RNA-binding protein A contains three copies of the double-stranded RNA-binding domain (dsRBD) in tandem arrangement. Two of them, xl1 and xl2, belong to the type A group of dsRBDs that show strong homologies to the entire length of a defined consensus sequence. The xl3 domain, in contrast, is a type B dsRBD which only matches the basic C-terminal end of the dsRBD consensus sequence. Here we show that only xl2 but neither xl1 nor xl3 are able to bind double-stranded RNA substrates in vitro, suggesting that different dsRBD copies have varying RNA binding activities. By fine mapping mutagenesis of the isolated xl2 domain, we identified at least two central aromatic amino acids and a C-terminal alpha-helix that are indispensable for dsRNA binding. Furthermore, we show that different charge distributions within the C-terminal alpha-helices of xl1 and xl2 seem responsible for the different RNA binding behaviors of these two dsRBDs. Analyses of the RNA binding properties of constructs containing various combinations of different dsRBDs reveal that type A dsRBDs exhibit a cooperative binding effect, whereas type B dsRBDs show a rather low binding activity, thus contributing only to a minor extent to a stable RNA-protein interaction.
Department of Cytology and Genetics, Institute of Botany, University of Vienna, Rennweg 14, 1030 Vienna, Austria.
B C
BC
Krovat
M F
MF
Jantsch
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D015152:Blotting, Northern; D015153:Blotting, Western; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D012330:RNA, Double-Stranded / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D012330:RNA, Double-Stranded; D016601:RNA-Binding Proteins