Arginine-rich motifs present multiple interfaces for specific binding by RNA

Abstract

A number of proteins containing arginine-rich motifs (ARMs) are known to bind RNA and are involved in regulating RNA processing in viruses and cells. Using automated selection methods we have generated a number of aptamers against ARM peptides from various natural proteins. Aptamers bind tightly to their cognate ARMs, with K(d) values in the nanomolar range, and frequently show no propensity to bind to other ARMs or even to single amino acid variants of the cognate ARM. However, at least some anti-ARM aptamers can cross-recognize a limited set of other ARMs, just as natural RNA-binding sites have been shown to exhibit so-called "chameleonism." We expand upon the number of examples of cross-recognition and, using mutational and circular dichroism (CD) analyses, demonstrate that there are multiple mechanisms by which RNA ligands can cross-recognize ARMs. These studies support a model in which individual arginine residues govern binding to an RNA ligand, and the inherent flexibility of the peptide backbone may make it possible for "semi-specific" recognition of a discrete set of RNAs by a discrete set of ARM peptides and proteins.

FIGURE 1.

Nondenaturing gel shifts for anti-Rev aptamer 5. Limiting aptamer RNA was incubated with various concentrations (shown in nanomolar) of (A,B) Rev peptide (broad and narrow concentration ranges, respectively), (D) RevN7D, and (E) RevR10Y peptides. Cross-recognitions (C) were assessed with 1024 nM of each peptide.

FIGURE 2.

Circular dichroism spectra. Spectra are grouped according to peptide binding to the (A) anti-Rex aptamer, (B) anti-Rev aptamer 5, (C) anti-RevN7D aptamer, (D) anti-CCMV Gag aptamer, and (E) anti-BMV Gag aptamer. Spectra are shown as the difference between the complex and the RNA alone.