xPat1 proteins bind RNA in vitro via a central region. (A) xPat1 proteins bind RNA homopolymers in vitro. Autoradiograph of in vitro–translated and 35[S]-Met–labeled xPat1a, xPat1b, hPat1b, and CPEB (control) bound to poly(A), poly(U), poly(C), poly(G), and Sepharose beads (B, as a control for unspecific binding). (B) xPat1a and xPat1b poly(G) binding is competed by wild-type, but not mutant, NRAS G-quadruplex–forming RNAs (Kumari et al. 2007). Approximately 5, 10, 20, and 40 molar equivalents of NRAS relative to poly(G) homopolymer were used as competitors, and the levels of Pat1 proteins resistant to competition were quantitated by densitometry. (C) xPat1 proteins bind RNA via region III (RIII). Graph of the percentage binding of different regions of xPat1 proteins and CPEB to the RNA homopolymers. (D) Helix x in region III is required for RNA binding. Graph of the percentage of binding in full length (FL), constructs lacking RIII (ΔRIII), or helix x (Δhelix x) bound to poly(U) and poly(G) homopolymers. Three independent experiments were performed and standard error bars are shown (C,D).
