RNA-Centric Methods: Toward the Interactome of Specific RNA Transcripts

Abstract

RNA-protein interactions play an important role in numerous cellular processes in health and disease. In recent years, the global RNA-bound proteome has been extensively studied, uncovering many previously unknown RNA-binding proteins. However, little is known about which particular proteins bind to which specific RNA transcript. In this review, we provide an overview of methods to identify RNA-protein interactions, with a particular focus on strategies that provide insights into the interactome of specific RNA transcripts. Finally, we discuss challenges and future directions, including the potential of CRISPR-RNA targeting systems to investigate endogenous RNA-protein interactions. Importance of Studying RNA-Protein ComplexesThe various types of RNA, such as mRNAs, rRNAs, and long noncoding (lnc)RNAs, are involved in a multitude of cellular processes. The classical view of RNAs serving merely as a template for protein synthesis has been expanded to catalytic, structural, and regulatory functions [1,2]. RNAs interact with RNA-binding proteins (RBPs; see Glossary), which play an essential role in RNA fate and function, such as in mRNA processing and translation [3]. In addition, RNA binding can also change the fate and function of proteins, for example by regulating protein stability and localization [4,5]. The importance of these interactions also becomes clear in the context of diseases that are characterized by perturbed RNA-protein interactions. Any alteration in expression level, structure, localization, or modification of either RNA or protein may disrupt these interactions and result in deregulation of the involved cellular processes [6,7]. For example, the RNA modification N6-methyladenosine (m6A) affects protein binding, and alterations in m6A levels are frequently observed in cancer [8][9][10].To characterize RNA-protein interactions, a rapidly expanding toolbox of methods is available, often classified into protein-centric and RNA-centric methods. Protein-centric methods, such as crosslinking immunoprecipitation (CLIP), rely on immunoprecipitation of the protein of interest followed by sequencing of the associated RNAs [11,12]. Conversely, RNA-centric methods rely on isolation of proteins associated with the RNA of interest. These methods led to the identification of many RBPs, including unexpected proteins, such as metabolic enzymes and transcription factors (reviewed in [5]). In this review, we provide an overview of the current and emerging RNAcentric approaches (Figure 1). In addition, we highlight the advantages, disadvantages, and challenges that have to be overcome to improve current methods. Furthermore, we discuss potential technical innovations to provide robust insights into endogenous protein-RNA interactomes. Global RNA-Binding ProteomeTo gain insight into the function of RNA-protein interactions, several methods have been developed in recent years to identify the global RNA-bound proteome. These methods preserve cellular RNA-protein interactions by crosslinking, which is then followed by c...

Opportunities and Challenges in Global Quantification of RNA-Protein Interaction via UV Cross-Linking

“…oligonucleotide probes are sensitive to modifications of the RNA sequence that might impair hybridization (Gräwe et al, 2020).…”

“…CLIP-like methods provide a detailed picture of the RBP-RNA interactome with single nucleotide resolution for a specific RBP of interest ( Hafner et al, 2010 ; König et al, 2010 ). Conversely, RNA-centric methods like RNA-interactome capture (RIC) use mass spectrometry-based proteomics to identify the RNA-bound proteome following biochemical isolation of RNAs ( Gebauer et al, 2020 ; Gräwe et al, 2020 ). In analogy to CLIP, amino acid resolution of RBP-RNA interactions can be obtained ( Kramer et al, 2014 ; Bae et al, 2020 ).…”

“…All of these methods can be categorized as CLIP- or RIC-like depending on the readout (transcriptomics or proteomics, respectively). For a detailed methodological discussion, we refer the interested reader to excellent reviews on the available methods and their limitations ( Ramanathan et al, 2019 ; Gebauer et al, 2020 ; Gräwe et al, 2020 ; Smith et al, 2020 ).…”

“…In CLIP-like assays, the pull-down efficiency of RBPs (usually with antibodies) is generally assumed to be independent of the bound RNA sequences. In RIC-like assays on the other hand, the pull-down efficiency of RBPs strongly depends on the isolation efficiency of their RNA targets ( Gräwe et al, 2020 ). Features such as RNA length, subcellular localization, base composition, modifications and secondary structures can all influence RNA isolation.…”

“…For instance, enrichment of the RBP-RNA complex using organic phase separation isolates complexes bound to RNAs as small as 30 nucleotides, but isolation efficiency drops dramatically for smaller RNAs ( Urdaneta et al, 2019 ). Also, methods that enrich specific RNAs via hybridization to complementary oligonucleotide probes are sensitive to modifications of the RNA sequence that might impair hybridization ( Gräwe et al, 2020 ).…”