Engineering RNA sequence specificity of Pumilio repeats

Abstract

Puf proteins bind RNA sequence specifically and regulate translation and stability of target mRNAs. A "code" for RNA recognition has been deduced from crystal structures of the Puf protein, human Pumilio1, where each of eight repeats binds an RNA base via a combination of three side chains at conserved positions. Here, we report the creation of seven soluble mutant proteins with predictably altered sequence specificity, including one that binds tightly to adenosine-uracil-rich element RNA. These data show that Pumilio1 can be used as a scaffold to engineer RNA-binding proteins with designed sequence specificity.

Fig. 1.

Recognition of RNA by HsPUM1-HD. (a) Schematic representation of RNA:protein interaction between HsPUM1-HD and NRE RNA (13). Protein repeats are indicated by squares and RNA bases by ovals (dashed lines, hydrogen bonds; parentheses, van der Waals contacts). Blue, green, and purple side chains, respectively, are in equivalent positions in each repeat. (b) Interaction of HsPUM1-HD with a uracil (Top), guanine (Middle), or adenine (Bottom). The RNA and side chains that contact the RNA are shown in stick representation (dark blue, nitrogen; red, oxygen; yellow, light blue, green, or purple, carbon). PDB ID code is 1M8Y.

Fig. 2.

Analysis of RNA binding for mutant HsPUM1-HD proteins. Representative analyses of equilibrium binding data for MUT7-2 protein and G2U RNA (a), MUT6-2/7-2 protein and GU23UG RNA (b), MUT1-1 protein and A8G RNA (c), MUT3-2 protein and A6G RNA (d), MUT3-1 protein and A6U RNA (e), and MUT7-2/3-1 protein and ARE RNA (f). Binding to cognate RNA is red and to wild-type RNA is black. These analyses assume one HsPUM1-HD binds to one RNA molecule.

Fig. 3.

Analysis of competitive RNA binding for mutant HsPUM1-HD proteins. Representative analyses of equilibrium-binding competition data for MUT7-2 protein (a), MUT6-2/7-2 protein (b), and MUT7-2/3-1 protein (c). Competition of binding of mutant protein to cognate mutant RNA with unlabeled wild-type RNA is black and with unlabeled cognate RNA is red. (d) A representative EMSA of an equilibrium-binding competition for MUT7-2 protein. Labeled cognate mutant RNA was competed with unlabeled cognate mutant RNA. The concentrations of unlabeled competitor RNA are indicated under each lane in the gel.