Comparative Mutational Analysis of the Double-stranded RNA Binding Domains of Xenopus laevis RNA-binding Protein A

Abstract

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.

Hox Transcription Factor Ultrabithorax Ib Physically and Genetically Interacts with Disconnected Interacting Protein 1, a Double-stranded RNA-binding Protein

“…10 (lanes 12 and 13), Ubx Ib binds DNA with a single consensus binding site of 5Ј-TAATGG-3Ј (57) to generate a single shifted band. Under the same conditions, only a small degree of DIP1-c⅐DNA binding was observed, consistent with the general ability of dsRBDs to weakly interact with dsDNA (37,38,50). However, when DIP1-c and Ubx Ib are combined, a novel DNA complex is observed.…”

“…Importantly, two dsRBDs (residues 171-237 and 335-403) (50) were also identified in DIP1. dsRBDs are 65-68-amino acid motifs that are divided into two categories: type A domains, which are conserved throughout the motif in sequence and secondary structure of ␣-␤-␤-␤-␣ (where ␣ denotes ␣-helix and ␤ is ␤-sheet), and type B domains, which are only conserved in the C-terminal helix (37,50). Interestingly, the first dsRBD of DIP1 is a type A domain whereas the second dsRBD is a type B domain (data not shown) (24).…”

“…6 and Table I). Strong interaction with dsRNA and weak interaction with single-stranded RNA and dsDNA is characteristic of proteins containing double-stranded RNA-binding domains (37,38,50). Therefore, the double-stranded RNA-binding domains identified through sequence similarity are functionally active.…”

Solution structure of the N-terminal dsRBD of Drosophila ADAR and interaction studies with RNA

“…This situation is not unique to dADAR dsRBD1, as the stacking of positively charged residues equivalent to K110 and R114 (dADAR dsRBD1 residue numbering - Figure 3E) over conserved aromatic rings, namely Y78 for K110 and F92 for R114, allows these long side chains to adopt an elongated conformation ( Figure 6). It has been proposed earlier that these aromatic side chains would therefore be essential for proper orientation of the long positively charged residues within the KKxAK consensus motif at the N-terminal tip of helix α2 [32], and their importance for the dsRNA binding capacity of dsRBDs have been also demonstrated [30,33,56]. Surprisingly, the side chain of K111 also presents an elongated conformation in the free form which in this case cannot be explained by a stacking onto an aromatic residue.…”

Intact RNA-binding Domains Are Necessary for Structure-specific DNA Binding and Transcription Control by CBTF122 during Xenopus Development

“…The NMR structure of the Staufen dsRBD has been solved (23), as has the crystal structure of the Xlrbpa dsRBD complexed with dsRNA (24). Saturation mutagenesis studies on the dsRBD of Xlrbpa demonstrated that the substitution of a conserved central Phe residue by Ala in both Staufen and Xlrbpa dsRBDs leads to abolition of RNA binding activity (23,25). Reference to the published structure of the dsRBD-RNA complex demonstrates that this residue is important in stabilizing a lysine side chain that makes important backbone contacts adjacent to the major groove (24).…”