Review
Global Approaches in Studying RNA-Binding Protein Interaction Networks

https://doi.org/10.1016/j.tibs.2020.03.005Get rights and content

Highlights

  • Higher-order interactions between RNA binding proteins (RBPs) on specific mRNAs are commonly found to mediate complex regulatory outcomes.

  • Transcriptome-wide methods and integrative analyses have been developed to probe RBP binding specificity, in vivo RNA targets, and protein partners, as well as measure RBP impact on RNA levels, structure, and translation.

  • Meta-analyses of these large datasets identify common principles of hierarchy, autoregulation, and functional coordination of RBPs binding mRNAs.

  • RNAs and RBPs simultaneously engage in a spectrum of interaction types, from weak and low-specificity interactions that drive phase separation, to the stronger, more binary interactions that impart distinct regulatory fates on specific mRNAs.

RNA-binding proteins (RBPs) play crucial roles in almost all aspects of cellular biology. RBP binding at specific target sites impacts expression of functionally coordinated sets of mRNAs and involves combinatorial and dynamic interactions with other RBPs. The complexity and principles of these regulatory networks are only beginning to be understood. In recent years, transcriptome-wide experimental and computational methods to study RBPs and their interactions with RNA provided new insights into their function. Here, we review the approaches used in examining RBPs and their networks and the concepts that have been developed. We emphasize studies focusing on RBP–RNA interactions and higher-order RBP coregulation and describe approaches that integrate multiple types of transcriptome-wide data to form a global picture of these regulatory pathways.

Section snippets

From RNA-Binding Protein (RBP) Basics to RBP Networks

RBPs orchestrate most essential cellular processes. Some RBP–RNA interactions form stable ribonucleoprotein (RNP) (see Glossary) particles with defined roles, while other RBPs (and RNPs) interact transiently to process, regulate the function, and control the fate of virtually all RNAs in the cell. Importantly, RBPs are the key players in post-transcriptional regulation of both mRNAs and noncoding RNAs, encompassing RNA splicing, transport, modification, stability, and translation [1,2]. Many of

Approaches to Study Individual RBP Function

To illuminate the molecular mechanisms that underpin the cellular roles of RBPs, researchers have been systematically addressing several key questions about the binding and impact of individual RBPs and their interplay with the RNA landscape. For example, determining RBP effects on mRNA stability and splicing patterns is informative to their molecular modes of action. These measurements have typically involved knockdown/knockout/overexpression of an RBP of interest followed by RNA-sequencing

Tackling RBP–RBP Interactions

Finally, while the focus of most studies to date has been on the functional roles of individual RBPs, it is recognized that higher-order interactions between RBPs on specific mRNAs are common and these interactions mediate pervasive and complex regulatory outcomes. Throughout their lifecycles, mRNAs are continuously associated with large and dynamically changing sets of RBPs [2,47,48]. In addition, recent studies hint that RBPs form large regulatory networks that act to coordinate complex

Interaction Types and Mechanisms Gathered from Studies on Specific mRNAs

Examination of interactions between particular RBPs and/or miRNAs on specific mRNAs (typically using reporter assays, site-directed mutagenesis, manipulation of RBP levels, and co-IP) have delineated some of the mechanisms of such coregulatory events and their effects on expression outcomes, as detailed in recent reviews [54., 55., 56.]. The interactions can be cooperative, where the binding of one RBP leads to increased binding and/or activity of its partner RBP (for recent examples, see [57.,

Transcriptome-Wide Integrative Approaches to Detect and Characterize Interactions between RBPs

In recent years, global approaches have provided novel insights into the scope and complexity of combinatorial post-transcriptional regulation. These studies utilize various mixtures of computational and experimental methods, such as RNA-seq, RIP-chip/seq, CLIP-seq, and bioinformatics based on RBP-binding motifs, as described later (illustrated in Figure 3).

Meta-Analyses Support Higher-Order RBP Interaction Networks

Using the complex and potentially combinatorial RBP regulation on individual transcripts as a building block, RBPs establish broad regulatory networks with emerging principles of autoregulation, hierarchy, and functional coordination (Figure 4). Evidence for such interaction networks has been uncovered in large meta-analyses, where many global datasets characterizing RBP binding (typically from CLIP-seq experiments), RBP localization (by immunofluorescence), transcript abundance (from RNA-seq

Concluding Remarks

The RNA landscape is a crowded environment, making combinatorial interactions between RBPs and RNAs common. The overall regulatory interactions between RBPs and RNA span a spectrum of mechanisms and it is important to stress that RNAs and RBPs can simultaneously participate in several kinds of interactions and their combination dictates the full cellular response. On one side of the spectrum are the stronger, more binary interactions between RBPs and RNAs, which drive the binding at specific

Acknowledgments

The authors would like to thank members of the Karginov laboratory for stimulating discussion, as well as Dr Dorothee Dormann and Dr Saskia Hutten for comments on the manuscript.

Glossary

Alternative splicing (AS)
the process of producing different mature (spliced) transcript variants from the same pre-mRNA, mediated by RBPs that affect splice site selection.
AU-rich elements (AREs)
a type of RBP-binding motif, often found within the 3′ UTR; defined as a region with frequent adenine and uridine bases.
m6A
(N6-methyladenosine); methylation of the adenosine base at the nitrogen-6 position; an abundant internal modification in mRNA that affects its fate in several aspects.
Membrane-less

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