Mapping transcriptome-wide protein-RNA interactions to elucidate RNA regulatory programs

Abstract

Background: Our understanding of post-transcriptional gene regulation has increased exponentially with the development of robust methods to define protein-RNA interactions across the transcriptome. In this review, we highlight the evolution and successful applications of crosslinking and immunoprecipitation (CLIP) methods to interrogate protein-RNA interactions in a transcriptome-wide manner.

Results: Here, we survey the vast array of in vitro and in vivo approaches used to identify protein-RNA interactions, including but not limited to electrophoretic mobility shift assays, systematic evolution of ligands by exponential enrichment (SELEX), and RIP-seq. We particularly emphasize the advancement of CLIP technologies, and detail protocol improvements and computational tools used to analyze the output data. Importantly, we discuss how profiling protein-RNA interactions can delineate biological functions including splicing regulation, alternative polyadenylation, cytoplasmic decay substrates, and miRNA targets.

Conclusions: In summary, this review summarizes the benefits of characterizing RNA-protein networks to further understand the regulation of gene expression and disease pathogenesis. Our review comments on how future CLIP technologies can be adapted to address outstanding questions related to many aspects of RNA metabolism and further advance our understanding of RNA biology.

Keywords: CLIP; RNA binding proteins; RNA networks; post-transcriptional regulation.

Figure 1.. Integration of CLIP methods with computational analysis to explore protein-RNA interactions and their biological impact.

(A) Simplified schematic illustrating the CLIP workflow to generate transcriptome-side maps of RBP-RNA interactions occurring in vivo. (B) Integration of next generation sequencing with CLIP data can be used to identify RBPs that function as (i) repressors of alternative splicing (AS), (ii) enhancers of alternative splicing, or (iii) regulators of alternative polyadenylation (APA). CLIP can also be performed on AGO proteins to map miRNA binding sites that downregulate target mRNAs (iv). (C) Application of CLIP methods to define RBP regulatory networks from position-dependent and combinatorial functions. CLIP profiles, in conjunction with high-throughput sequencing, can characterize transcript/protein diversity in specific cellular contexts, protein networks, localization patterns, translational fates of mRNAs, and substrates of mRNA decay. (D) Identification of complex protein-RNA interaction networks that regulate gene expression is critical to understanding human development and disease.