Target selection by natural and redesigned PUF proteins

Abstract

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.PUF proteins | RNA-binding proteins | synthetic biology | designer protein | CLIP-seq

The PUF binding landscape in metazoan germ cells

“…The greater number and diversity of FBF binding motifs identified in this work may enable fine-tuning of FBF-RNA interactions. Moreover, recent evidence from yeast indicates that PUF binding at short motif elements has small repressive effects (∼2%) on target mRNAs, suggesting subtle regulatory effects from binding, even at alternate motifs (Porter et al 2015). Second, most FBF binding sites are in the 3 ′ UTR and are enriched toward the terminal end of those UTRs.…”

“…Single RNA binding proteins can regulate 100s to 1000s of RNAs with many targets having related functions, which enables coordinated biological control (Keene and Tenenbaum 2002;Gerber et al 2004;Darnell 2010;Weyn-Vanhentenryck et al 2014;Hogan et al 2015;Kershaw et al 2015;Lapointe et al 2015;Porter et al 2015;Wilinski et al 2015). Understanding RNA regulatory networks in metazoans requires knowing which RNAs are regulated and how they are recognized in their native context.…”

Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

“…HuR (ELAVL1) has been shown to interact with mRNA and pre-mRNA in several CLIP studies and has a well-documented binding motif (5'-UUUUUU -3') (26). After in vivo labeling of cellular RNA using 4-thiouridine (4SU) and UV irradiation at 365 nm, we performed i) classical PAR-CLIP analysis (PAR-CLIPclassic) (11,27) of HuR, ii) a PAR-CLIP variant using onbead ligation of adapters (PAR-CLIP-on-beads) (28,29), and iii) a version in which we use phenol extraction (termed pCLIP) for removal of unbound RNA instead of PAGE/membrane excision (Fig. 4a).…”

“…As an alternative, the ligation of the 3´/ 5´adapters can be achieved directly on the beads used in the affinity capture of the selected clRNP (28,29). Onbeads adapters ligation was done by incubating the FLAG-clHuR-RNA beads with the 3´adapter in the presence of Rnl2(1-249)K227Q ligase and PEG-8000 overnight at 4°C.…”

Harnessing short poly(A)-binding protein-interacting peptides for the suppression of nonsense-mediated mRNA decay

“…So far, we have only demonstrated the activity of PAM2 motifs by tethered function analysis. However, PAM2 peptides also may be combined with sequence-specific RNA-binding molecules, such as Pumilio homology domains5051, CRISPR-Cas95253 or antisense oligonucleotides54 to suppress NMD independent of tethering. Applied in combination with read-through drugs, PAM2 peptides may also enable to restore the expression of nonsense-mutated genes.…”