Nested PUF Proteins: Extending Target RNA Elements for Gene Regulation.

RNA-binding proteins recognizing unique sequences within large transcriptomes serve as a powerful tool to control RNA metabolism. Pumilio and fem-3 mRNA-binding factor (PUF) proteins are considered good candidates for such tools, because they are typically composed of eight highly homologous repeat segments and can be designed to recognize arbitrary 8 nt RNA sequences. However, a specific 8 nt RNA sequence is found at multiple sites in various RNAs in the transcriptome, making it difficult to specifically target a single RNA. Designer PUF proteins recognizing longer RNA sequences should achieve more selective binding. Here, we propose an approach for creating 16-repeat PUFs capable of targeting a single, unique mRNA in the transcriptome. Our design is simple and involves either the tandem alignment of two PUF segments or the nesting of one PUF segment within another. Designed 16-repeat PUFs bound to the target RNA sequence without partial recognition derived from the original 8-repeat PUF. Furthermore, based on our strategy, expression of an endogenous mRNA was selectively and effectively modulated, demonstrating the applicability of 16-repeat PUF proteins for regulating endogenous RNA metabolism.