Engineering RNA-binding proteins

RNA-protein interactions have key roles in the regulation of gene expression and are vital for many cellular processes and complex developmental programs in eukaryotes. Proteins that have the ability to bind RNAs tend to do so in various modes that are often difficult to predict, limiting the ability to engineer these RNA-binding proteins for medical and biotechnological use. Hence, engineering proteins that can bind a specific RNA sequence has many potential applications, analogous to that of siRNAs and miRNAs, with even more potential benefits. The ability to fuse RNA-binding proteins to any desired effector domain, in turn enabling the manipulation of any aspect of the target RNA's metabolism makes engineering these proteins highly appealing. Here, the recognition of PUF repeats beyond adenine, guanine and uracil has been achieved through directed evolution, enabling them to specifically bind cytosine. With this code, PUF domains capable of selectively binding RNA targets of diverse sequence and structure can be designed. Unlike the PUFs, the basis for nucleotide RNA recognition by pentatricopeptide repeat (PPR) proteins, another RNA-binding protein, remains ambiguous. Here, computational methods have been used to create a stable, highly reduced PPR architecture for the study of RNA-binding specificity and the design of specific tools to manipulate RNA metabolism. We used these synthetic PPRs to examine the amino acid codes for nucleotide recognition by PPRs, which also revealed that PPRs have a modular recognition mechanism similar to that of PUFs. These findings provide a significant step towards predicting native binding sites of the vast number of PPR proteins found in nature. It also highlights the possibility of a PPR scaffold to be engineered for new functions and sequence specificities.