A computationally-enhanced hiCLIP atlas reveals Staufen1 RNA binding features and links 3’ UTR structure to RNA metabolism

Abstract

The structure of mRNA molecules plays an important role in its interactions with trans-acting factors, notably RNA binding proteins (RBPs), thus contributing to the functional consequences of this interplay. However, current transcriptome-wide experimental methods to chart these interactions are limited by their poor sensitivity. Here we extend the hiCLIP atlas of duplexes bound by Staufen1 (STAU1) ~10-fold, through careful consideration of experimental assumptions, and the development of bespoke computational methods which we apply to existing data. We present Tosca, a Nextflow computational pipeline for the processing, analysis and visualisation of proximity ligation sequencing data generally. We use our extended duplex atlas to discover insights into the RNA selectivity of STAU1, revealing the importance of structural symmetry and duplex-span-dependent nucleotide composition. Furthermore, we identify heterogeneity in the relationship between STAU1-bound 3' UTRs and metabolism of the associated RNAs that we relate to RNA structure: transcripts with short-range proximal 3' UTR duplexes have high degradation rates, but those with long-range duplexes have low rates. Overall, our work enables the integrative analysis of proximity ligation data delivering insights into specific features and effects of RBP-RNA structure interactions.

The role of secondary structures in the functioning of 3′ untranslated regions of mRNA

“…[ 6 ] A recent study using the hiCLIP method expanded the atlas of mRNA duplexes that bind to STAU1 by approximately 10‐fold, with 80.1% of the binding sites located in the 3′ UTR. [ 76 ] Staufen proteins are involved in multiple processes, participating in mRNA transport, translational regulation and SMD depending on spatio‐temporal context. The mechanism for switching between these functions is unclear, but it depends on regions of mRNA bound by Staufen (5′ UTR, coding sequence or 3′ UTR) and protein partners interacting with Staufen in each case.…”

lhCLIP reveals the in vivo RNA–RNA interactions recognized by hnRNPK

“…However, there is still a need for strategies to identify RNA interactions associated with specific RBPs. CLASH (crosslinking, ligation, and sequencing of hybrids) [8,9] and hiCLIP (RNA hybrid and individual-nucleotide resolution ultraviolet crosslinking and immunoprecipitation) [10] have been developed to detect RNA duplexes bound by specific RBPs. However, both techniques require introduction of exogenous expression of the target protein and can only provide chimeric read coverage of approximately 2%.…”

“…However, both techniques require introduction of exogenous expression of the target protein and can only provide chimeric read coverage of approximately 2%. Recently, the hiCLIP atlas of duplexes bound by Staufen1(STAU1) has been extended by approximate-ly10-fold through the development of bespoke computational methods which were apply to existing data [10]. Additionally, a novel method called captured RICseq (CRIC-seq) has been developed to profile the RNA interactome arranged by a single RBP [11].…”