Noncoding RNA sequences, including long noncoding RNAs, small nucleolar RNAs, and untranslated mRNA regions, accomplish many of their diverse functions through direct interactions with RNA-binding proteins (RBPs). Recent efforts have identified hundreds of new RBPs that lack known RNA-binding domains, thus underscoring the complexity and diversity of RNA-protein complexes. Recent progress has expanded the number of methods for studying RNAprotein interactions in two general categories: approaches that characterize proteins bound to an RNA of interest (RNA-centric), and those that examine RNAs bound to a protein of interest (protein-centric). Each method has unique strengths and limitations, which makes it important to select optimal approaches for the biological question being addressed. Here we review methods for the study of RNA-protein interactions, with a focus on their suitability for specific applications. RNA and proteins are interconnected biomolecules that can promote each other's life cycles and functions through physical interactions 1. The coding sequence of mRNA carries the instructions for protein synthesis and some regulatory sequences, and the untranslated regions of mRNA influence the fate of the encoded protein by regulating its protein translation, localization, and interactions with other proteins 2. Proteins, in turn, can bind and modulate RNA expression and function from RNA synthesis to degradation 3. RNA-protein interactions are key to cellular homeostasis, and perturbations of RNA-RBP interactions can lead to cellular dysfunction and disease 4,5. Recent work has substantially expanded the number of putative RNA-protein associations in eukaryotes, underscoring the need for a versatile array of methods to identify and characterize their interactions 6,7. Methods for studying the physical interactions between RNA and protein can be classified by the type of molecule they start with. RNA-centric methods start with an RNA of interest

PUF (Pumilio/FBF) proteins are RNA-binding proteins and conserved stem cell regulators. The Caenorhabditis elegans PUF proteins FBF-1 and FBF-2 (collectively FBF) regulate mRNAs in germ cells. Without FBF, adult germlines lose all stem cells. A major gap in our understanding of PUF proteins, including FBF, is a global view of their binding sites in their native context (i.e., their "binding landscape"). To understand the interactions underlying FBF function, we used iCLIP (individual-nucleotide resolution UV crosslinking and immunoprecipitation) to determine binding landscapes of C. elegans FBF-1 and FBF-2 in the germline tissue of intact animals. Multiple iCLIP peak-calling methods were compared to maximize identification of both established FBF binding sites and positive control target mRNAs in our iCLIP data. We discovered that FBF-1 and FBF-2 bind to RNAs through canonical as well as alternate motifs. We also analyzed crosslinking-induced mutations to map binding sites precisely and to identify key nucleotides that may be critical for FBF-RNA interactions. FBF-1 and FBF-2 can bind sites in the 5 ′ UTR, coding region, or 3 ′ UTR, but have a strong bias for the 3 ′ end of transcripts. FBF-1 and FBF-2 have strongly overlapping target profiles, including mRNAs and noncoding RNAs. From a statistically robust list of 1404 common FBF targets, 847 were previously unknown, 154 were related to cell cycle regulation, three were lincRNAs, and 335 were shared with the human PUF protein PUM2.

Ribonucleotidyl transferases (rNTases) add non-templated ribonucleotides to diverse RNAs. We developed TRAID-Seq, a screening strategy in S. cerevisiae to identify sequences added to a reporter RNA at single-nucleotide resolution by overexpressing candidate enzymes from different organisms. The rNTase activities of 22 previously unexplored enzymes were determined. In addition to poly(A)- and poly(U)-adding enzymes, we identified a C-adding enzyme that is likely part of a two-enzyme system that adds CCA to tRNAs in a eukaryote; a nucleotidyl transferase that adds nucleotides to RNA without apparent nucleotide preference; and a poly(UG) polymerase, C. elegans MUT-2, which adds alternating U and G nucleotides to form poly(UG) tails. MUT-2 is known to be required for certain forms of RNA silencing, and mutations in the enzyme that are defective in silencing fail to add poly(UG) tails in our assay. We propose that MUT-2 poly(UG) polymerase activity is required to promote genome integrity and RNA silencing.

mRNA control hinges on the specificity and affinity of proteins for their RNA binding sites. Regulatory proteins must bind their own sites and reject even closely related noncognate sites. In the PUF [Pumilio and fem-3 binding factor (FBF)] family of RNA binding proteins, individual proteins discriminate differences in the length and sequence of binding sites, allowing each PUF to bind a distinct battery of mRNAs. Here, we show that despite these differences, the pattern of RNA interactions is conserved among PUF proteins: the two ends of the PUF protein make critical contacts with the two ends of the RNA sites. Despite this conserved "two-handed" pattern of recognition, the RNA sequence is flexible. Among the binding sites of yeast Puf4p, RNA sequence dictates the pattern in which RNA bases are flipped away from the binding surface of the protein. Small differences in RNA sequence allow new modes of control, recruiting Puf5p in addition to Puf4p to a single site. This embedded information adds a new layer of biological meaning to the connections between RNA targets and PUF proteins.mRNA turnover | RNA regulation | translation | 3′UTR elements

C. elegans germline stem cells exist within a stem cell pool that is maintained by a single-celled mesenchymal niche and Notch signaling. Downstream of Notch signaling, a regulatory network governs stem cells and differentiation. Central to that network is the FBF RNA-binding protein, a member of the widely conserved PUF family that functions by either of two broadly conserved mechanisms to repress its target mRNAs. Without FBF, germline stem cells do not proliferate and they do not maintain their naïve, undifferentiated state. Therefore, FBF is a pivotal regulator of germline self-renewal. Validated FBF targets include several key differentiation regulators as well as a major cell cycle regulator. A genomic analysis identifies many other developmental and cell cycle regulators as likely FBF targets and suggests that FBF is a broad-spectrum regulator of the genome with>1,000 targets. A comparison of the FBF target list with similar lists for human PUF proteins, PUM1 and PUM2, reveals ∼200 shared targets. The FBF hub works within a network controlling self-renewal vs. differentiation. This network consists of classical developmental cell fate regulators and classical cell cycle regulators. Recent results have begun to integrate developmental and cell cycle regulation within the network. The molecular dynamics of the network remain a challenge for the future, but models are proposed. We suggest that molecular controls of C. elegans germline stem cells provide an important model for controls of stem cells more broadly.

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

Protein-protein interactions between domains within fatty acid and polyketide synthases are critical to catalysis, but their contributions remain incompletely characterized. A practical, quantitative system for establishing functional interactions between modifying enzymes and the acyl carrier protein that tethers the nascent polymer would offer a valuable tool for understanding and engineering these enzyme systems. Mechanism-based crosslinking of modular domains offers a potential diagnostic to highlight selective interactions between modular pairs. Here kinetic activity analysis and isothermal titration calorimetry are shown to correlate the efficiency of a ketosynthase-carrier protein crosslinking method to the binding affinity and transacylase activity that occurs in ketosynthase chain elongation.

Quantitative criteria to identify proteins as RNA-binding proteins (RBPs) are presently lacking, as are criteria to define RBP target RNAs. Here, we develop an ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing method, easyCLIP. easyCLIP provides absolute cross-link rates, as well as increased simplicity, efficiency, and capacity to visualize RNA libraries during sequencing library preparation. Measurement of >200 independent cross-link experiments across >35 proteins identifies an RNA cross-link rate threshold that distinguishes RBPs from non-RBPs and defines target RNAs as those with a complex frequency unlikely for a random protein. We apply easyCLIP to the 33 most recurrent cancer mutations across 28 RBPs, finding increased RNA binding per RBP molecule for KHDRBS2 R168C, A1CF E34K and PCBP1 L100P/Q cancer mutations. Quantitating RBP-RNA interactions can thus nominate proteins as RBPs and define the impact of specific disease-associated RBP mutations on RNA association.