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Nested PUF Proteins: Extending Target RNA Elements for Gene Regulation

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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… Click to show full abstract

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.

Keywords: puf proteins; puf; repeat; target rna

Journal Title: ChemBioChem
Year Published: 2018

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