Understanding and engineering RNA sequence specificity of PUF proteins

Abstract

PUF proteins are RNA-binding proteins named for founding members PUMILIO and fem-3 binding factor (FBF). Together these proteins represent the range of known RNA recognition properties. PUMILIO is a prototypical PUF protein whose RNA sequence specificity is simple, elegant, and predictable. FBF displays differences in RNA recognition that represent divergence from the prototype. Here we review recent studies that examine the engineering of sequence specificity of PUF proteins and its applications as well as studies that increase our understanding of the natural diversity of RNA recognition by this family of proteins.

Figure 1

Prototypical PUF protein RNA recognition by PUM1. (a) Crystal structure of PUM1 in complex with hb NRE RNA. Repeats are colored alternately blue and yellow and side chains that contact the RNA are shown. RNA and PUM1 side chains are colored by atom type (blue, nitrogen; red, oxygen; grey, carbon; orange, phosphorous; yellow, sulfate). Side chains that contact the edges of bases are shown with blue or yellow carbon atoms, and those that form stacking interactions are shown with magenta carbon atoms. (b) PUM1 RNA recognition code. Schematic representation of recognition of uracil, adenosine and guanine by PUM1 is shown. Recognition of cytosine cannot yet be engineered, but a single cytosine can be tolerated at the 5^th position in a recognition sequence. Dotted lines indicate hydrogen bonds, )))) indicates van der Waals contacts, and |||| indicates stacking interactions.

Figure 2

Applications of designed PUF proteins. (a) Tracking of endogenous RNA by fusing designed PUM1 with fluorescent protein (GFP). An N-terminal fragment of GFP (green) is fused at the N-terminus of PUM1 (PUM1-N) and a C-terminal fragment of GFP (yellow) is fused at the C-terminus of a second molecule of PUM1 (PUM1-C). Binding of both molecules to the target RNA allows reconstitution of GFP fluorescence. (b) Understanding FBF RNA recognition by design of repeat specificity. Schematic representations of two possible models of FBF:9-nt RNA interaction are shown at the top of the figure. Side chains that likely contact the RNA bases are shown. Model 1 shows the 5^th base (yellow) accommodated in the center of the protein while Model 2 shows the 9^th base (yellow) accommodated near the N terminus of the protein. Model 2 requires non-canonical repeat:base interactions, some of which were observed in structures of PUM1 by Gupta, et al. [20]. One example of how site-directed mutagenesis was used to probe FBF RNA interaction is shown in the bottom figure. Opperman, et al. [10] mutated repeat 2 to recognize a guanine (orange) and found that the mutant protein preferred the sequence A₇G₈A₉ (bracketed sequence) as predicted by Model 1 (Model 2 predicts G₇U₈A₉, which was not bound by the mutant FBF).

Figure 3

Accommodation of an ‘extra’ RNA base by PUF4 and PUM1. (a) Crystal structure of PUF4 in complex with HO target RNA reveals that the 7^th base is flipped away from the RNA-binding surface. This “flipping-out” of U₇ is stabilized by Arg651 of repeat 3, which stacks between bases A₆ and U₈ and directly contacts the backbone ribose of U₇. (b) Crystal structure of PUF4 T650C/C724R mutant demonstrates that Arg724 stacks between bases 4 and 5. (c) Crystal structure of PUM1 in complex with HO PUF5-BS RNA. The 6^th base is flipped away from the RNA-binding surface to best accommodate the sequence. Tyr1005 in repeat 5 and Gln968 in repeat 4 stabilize the displaced U₆ through van der Waals interactions with its phosphate and ribose groups, respectively. Atoms are colored as in Figure 1a.