Directed evolution of orthogonal RNA–RBP pairs through library-vs-library in vitro selection

RNA-binding proteins (RBPs) and their RNA ligands play many critical roles in gene regulation and RNA processing in cells. They are also useful for various applications in cell biology and synthetic biology. However, re-engineering novel and orthogonal RNA–RBP pairs from natural components remains challenging while such synthetic RNA–RBP pairs could significantly expand the RNA–RBP toolbox for various applications. Here, we report a novel library-vs-library in vitro selection strategy based on Phage Display coupled with Systematic Evolution of Ligands by EXponential enrichment (PD-SELEX). Starting with pools of 1.1 × 10¹² unique RNA sequences and 4.0 × 10⁸ unique phage-displayed L7Ae-scaffold (LS) proteins, we selected RNA–RBP complexes through a two-step affinity purification process. After six rounds of library-vs-library selection, the selected RNAs and LS proteins were analyzed by next-generation sequencing (NGS). Further deconvolution of the enriched RNA and LS protein sequences revealed two synthetic and orthogonal RNA–RBP pairs that exhibit picomolar affinity and >4000-fold selectivity.     

New approaches to target RNA binding proteins

RNA binding proteins (RBPs) are a large and diverse class of proteins that regulate all aspects of RNA biology. As RBP dysregulation has been implicated in a number of human disorders, including cancers and neurodegenerative disease, small molecule chemical probes that target individual RBPs represent useful tools for deciphering RBP function and guiding the production of new therapeutics. While RBPs are often thought of as tough-to-drug, the discovery of a number of small molecules that target RBPs has spurred considerable recent interest in new strategies for RBP chemical probe discovery. Here we review current and emerging technologies for high throughput RBP-small molecule screening that we expect will help unlock the full therapeutic potential of this exciting protein class.     

RNA-binding proteins in neurological diseases

Emerging studies support that RNA-binding proteins(RBPs)play critical roles in human biology and pathogenesis.RBPs are essential players in RNA processing and metabolism,including pre-mRNA splicing,polyadenylation,transport,surveillance,mRNA localization,mRNA stability control,translational control and editing of various types of RNAs.Aberrant expression of and mutations in RBP genes affect various steps of RNA processing,altering target gene function.RBPs have been associated with various diseases,including neurological diseases.Here,we mainly focus on selected RNA-binding proteins including Nova-1/Nova-2,HuR/HuB/HuC/HuD,TDP-43,Fus,Rbfox1/Rbfox2,QKI and FMRP,discussing their function and roles in human diseases.     

Impact of RNA–Protein Interaction Modes on Translation Control: The Versatile Multidomain Protein Gemin5

The fate of cellular RNAs is largely dependent on their structural conformation, which determines the assembly of ribonucleoprotein (RNP) complexes. Consequently, RNA‐binding proteins (RBPs) play a pivotal role in the lifespan of RNAs. The advent of highly sensitive in cellulo approaches for studying RNPs reveals the presence of unprecedented RNA‐binding domains (RBDs). Likewise, the diversity of the RNA targets associated with a given RBP increases the code of RNA–protein interactions. Increasing evidence highlights the biological relevance of RNA conformation for recognition by specific RBPs and how this mutual interaction affects translation control. In particular, noncanonical RBDs present in proteins such as Gemin5, Roquin‐1, Staufen, and eIF3 eventually determine translation of selective targets. Collectively, recent studies on RBPs interacting with RNA in a structure‐dependent manner unveil new pathways for gene expression regulation, reinforcing the pivotal role of RNP complexes in genome decoding.     

Prevalent RNA recognition motif duplication in the human genome

The sequence-specific recognition of RNA by proteins is mediated through various RNA binding domains, with the RNA recognition motif (RRM) being the most frequent and present in >50% of RNA-binding proteins (RBPs). Many RBPs contain multiple RRMs, and it is unclear how each RRM contributes to the binding specificity of the entire protein. We found that RRMs within the same RBP (i.e., sibling RRMs) tend to have significantly higher similarity than expected by chance. Sibling RRM pairs from RBPs shared by multiple species tend to have lower similarity than those found only in a single species, suggesting that multiple RRMs within the same protein might arise from domain duplication followed by divergence through random mutations. This finding is exemplified by a recent RRM domain duplication in DAZ proteins and an ancient duplication in PABP proteins. Additionally, we found that different similarities between sibling RRMs are associated with distinct functions of an RBP and that the RBPs tend to contain repetitive sequences with low complexity. Taken together, this study suggests that the number of RBPs with multiple RRMs has expanded in mammals and that the multiple sibling RRMs may recognize similar target motifs in a cooperative manner.     

Ribonomic approaches to study the RNA-binding proteome

Gene expression is controlled through a complex interplay among mRNAs, non-coding RNAs and RNA-binding proteins (RBPs), which all assemble along with other RNA-associated factors in dynamic and functional ribonucleoprotein complexes (RNPs). To date, our understanding of RBPs is largely limited to proteins with known or predicted RNA-binding domains. However, various methods have been recently developed to capture an RNA of interest and comprehensively identify its associated RBPs. In this review, we discuss the RNA-affinity purification methods followed by mass spectrometry analysis (AP-MS); RBP screening within protein libraries and computational methods that can be used to study the RNA-binding proteome (RBPome).     

PRIESSTESS: interpretable,high-performing models of the sequence and structure preferences of RNA-binding proteins

Modelling both primary sequence and secondary structure preferences for RNA binding proteins (RBPs) remains an ongoing challenge. Current models use varied RNA structure representations and can be difficult to interpret and evaluate. To address these issues, we present a universal RNA motif-finding/scanning strategy, termed PRIESSTESS (Predictive RBP-RNA InterpretablE Sequence-Structure moTif regrESSion), that can be applied to diverse RNA binding datasets. PRIESSTESS identifies dozens of enriched RNA sequence and/or structure motifs that are subsequently reduced to a set of core motifs by logistic regression with LASSO regularization. Importantly, these core motifs are easily visualized and interpreted, and provide a measure of RBP secondary structure specificity. We used PRIESSTESS to interrogate new HTR-SELEX data for 23 RBPs with diverse RNA binding modes and captured known primary sequence and secondary structure preferences for each. Moreover, when applying PRIESSTESS to 144 RBPs across 202 RNA binding datasets, 75% showed an RNA secondary structure preference but only 10% had a preference besides unpaired bases, suggesting that most RBPs simply recognize the accessibility of primary sequences.     

Organic phase separation opens up new opportunities to interrogate the RNA-binding proteome

Protein–RNA interactions regulate all aspects of RNA metabolism and are crucial to the function of catalytic ribonucleoproteins. Until recently, the available technologies to capture RNA-bound proteins have been biased toward poly(A) RNA-binding proteins (RBPs) or involve molecular labeling, limiting their application. With the advent of organic–aqueous phase separation–based methods, we now have technologies that efficiently enrich the complete suite of RBPs and enable quantification of RBP dynamics. These flexible approaches to study RBPs and their bound RNA open up new research avenues for systems-level interrogation of protein–RNA interactions.