REPOSITORY > RESULTS

Abstract

RNA molecules dynamically interact with RNA-binding proteins (RBPs), which control various aspects of RNA fate, such as its processing, localisation, and stability. Intricate networks of protein-RNA interactions thereby regulate gene expression and have a profound effect on downstream cellular processes. Most RBPs recognise specific motifs on their bound RNAs, characterised by linear nucleotide sequences, RNA structures, RNA modifications, or combinatorial patterns of these features. By understanding how these features determine the specificity of protein-RNA interactions, we can gain valuable insights into the fundamental mechanisms that govern gene expression and cellular processes. As the first step, protein-RNA interactions are identified within cells by transcriptomic experiments such as crosslinking and immunoprecipitation (CLIP), which identifies RNA crosslink sites of selected RBPs. However, each method has its unique experimental biases, which creates challenges in separating biological from technical signal. For this purpose, we introduce the positionally-enriched k-mer analysis (PEKA), a computational approach for discovery of linear-sequence motifs enriched in the proximity of RNA crosslink sites identified by CLIP. PEKA implements steps to minimise the effects of technical biases in motif discovery and by visualising the motifs around crosslink sites facilitates the comparison between distinct CLIP datasets. Here, we apply PEKA for a comparative study of binding motifs that are enriched across an array of distinct RBPs and CLIP methods, to gain insights into general features underlying RBP specificity, such as the presence of intrinsically disordered regions (IDRs) and canonical RNA-binding domains, as well as the variations in technical artefacts and specificity between CLIP datasets.
Furthermore, we extended the application of PEKA and related computational approaches to investigate the dynamic regulation of LIN28A—an RBP essential for promoting the switch from naïve to primed cell fate in early embryonic development. In this process, LIN28A mediates the rapid decay of naïve-pluripotency factor mRNAs, but it was unclear how it selectively targets these mRNAs. Our findings show that selectivity is achieved by activating LIN28A through phosphorylation of its IDR, profoundly changing its RNA interactions. Upon phosphorylation, LIN28A converges to AU-rich 3′-UTR termini bound by the cytoplasmic poly(A)-binding proteins. mRNAs targeted for decay exhibited higher multivalency of AU-rich motifs and a greater accumulation of both LIN28A and poly(A)-binding proteins at their terminal regions. Given that dysregulation of LIN28A is linked to cancers and diverse growth conditions, a deeper understanding of its regulatory mechanisms is crucial. This work adds to that understanding and demonstrates the value of comparative CLIP analyses to elucidate methodological biases, determinants of RBP-binding specificity and gain functional insights into how RBPs regulate the specificity and dynamics of cellular processes.