Pumilio/fem-3 mRNA binding factor (PUF) proteins bind RNA with sequence specificity and modularity, and have become exemplary scaffolds in the reengineering of new RNA specificities. Here, we report the in vivo RNA binding sites of wild-type (WT) and reengineered forms of the PUF protein Saccharomyces cerevisiae Puf2p across the transcriptome. Puf2p defines an ancient protein family present throughout fungi, with divergent and distinctive PUF RNA binding domains, RNA-recognition motifs (RRMs), and prion regions. We identify sites in RNA bound to Puf2p in vivo by using two forms of UV cross-linking followed by immunopurification. The protein specifically binds more than 1,000 mRNAs, which contain multiple iterations of UAAU-binding elements. Regions outside the PUF domain, including the RRM, enhance discrimination among targets. Compensatory mutants reveal that one Puf2p molecule binds one UAAU sequence, and align the protein with the RNA site. Based on this architecture, we redesign Puf2p to bind UAAG and identify the targets of this reengineered PUF in vivo. The mutant protein finds its target site in 1,800 RNAs and yields a novel RNA network with a dramatic redistribution of binding elements. The mutant protein exhibits even greater RNA specificity than wild type. The redesigned protein decreases the abundance of RNAs in its redesigned network. These results suggest that reengineering using the PUF scaffold redirects and can even enhance specificity in vivo.
Keywords: CLIP-seq; PUF proteins; RNA-binding proteins; designer protein; synthetic biology.