Abstract
Identification of the protein domains that are responsible for RNA recognition has lagged behind the characterization of protein-DNA interactions. However, it is now becoming clear that a range of structural motifs bind to RNA and their structures and molecular mechanisms of action are beginning to be elucidated. In this report, we have expressed and purified one of the two putative RNA-binding domains from ZNF265, a protein that has been shown to bind to the spliceosomal components U1-70K and U2AF35 and to direct alternative splicing. We show that this domain, which contains four highly conserved cysteine residues, forms a stable, monomeric structure upon the addition of 1 molar eq of Zn(II). Determination of the solution structure of this domain reveals a conformation comprising two stacked beta-hairpins oriented at approximately 80 degrees to each other and sandwiching the zinc ion; the fold resembles the zinc ribbon class of zinc-binding domains, although with one less beta-strand than most members of the class. Analysis of the structure reveals a striking resemblance to known RNA-binding motifs in terms of the distribution of key surface residues responsible for making RNA contacts, despite a complete lack of structural homology. Furthermore, we have used an RNA gel shift assay to demonstrate that a single crossed finger domain from ZNF265 is capable of binding to an RNA message. Taken together, these results define a new RNA-binding motif and should provide insight into the functions of the >100 uncharacterized proteins in the sequence data bases that contain this domain.
Highlights
The zinc finger architecture has provided a diverse range of structures and functions (1), including recognition of protein, DNA, and RNA targets
The Structure of ZNF265-F1—The crossed finger domain seems to be a subclass of the zinc ribbon fold, and it adds to the already large family of zinc-binding domains in which the zinc probably serves only a structural role (1)
It is clear that these H/C-X2–5-H/C motifs take up a limited number of conformations, and presumably form a stable macrocyclic structure when bound to a zinc ion
Summary
Sequence Analysis—All sequence analysis and alignments were performed using the software pileup and prettybox (Australian Genome Information Service).. All NMR experiments used for determination of the structure of ZNF265-F1 were carried out at 298 K on a 600-MHz Bruker DRX600 spectrometer, equipped with a 5-mm triple resonance probe and three-. Structure calculations were performed in DYANA using a distance geometry/simulated annealing protocol (30) and additional NOEs were assigned iteratively based on earlier sets of structures. To include additional distance and angle constraints that maintain the tetrahedral bonding geometry and appropriate bond lengths with the zinc atom (35), as well as to incorporate ambiguous restraints, subsequent structural refinement was performed in CNS (36) using the package ARIA1.2 (37, 38). 0.1–5.0 g of affinity purified GSTZNF265-F1 protein was preincubated with 150 g of heparin (Sigma) for 10 min at room temperature in CEB buffer before addition of the RNA probe (200 cps). Samples were electrophoresed through a 4% native polyacrylamide gel and proteinRNA complexes were visualized by autoradiography
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