Matrix-screening reveals a vast potential for direct protein-protein interactions among RNA binding proteins

Abstract

RNA-binding proteins (RBPs) are crucial factors of post-transcriptional gene regulation and their modes of action are intensely investigated. At the center of attention are RNA motifs that guide where RBPs bind. However, sequence motifs are often poor predictors of RBP-RNA interactions in vivo. It is hence believed that many RBPs recognize RNAs as complexes, to increase specificity and regulatory possibilities. To probe the potential for complex formation among RBPs, we assembled a library of 978 mammalian RBPs and used rec-Y2H matrix screening to detect direct interactions between RBPs, sampling > 600 K interactions. We discovered 1994 new interactions and demonstrate that interacting RBPs bind RNAs adjacently in vivo. We further find that the mRNA binding region and motif preferences of RBPs deviate, depending on their adjacently binding interaction partners. Finally, we reveal novel RBP interaction networks among major RNA processing steps and show that splicing impairing RBP mutations observed in cancer rewire spliceosomal interaction networks. The dataset we provide will be a valuable resource for understanding the combinatorial interactions of RBPs with RNAs and the resulting regulatory outcomes.

Deep structural insights into RNA‐binding disordered protein regions

“…Spliceosomal networks are also highly sensitive to mutations in RNA binding regions, ordered and disordered alike. Mutations can lead to the remodeling of the complete spliceosomal network (Lang et al, 2021), or can cause the abnormal localization of the protein (Gaertner et al, 2020), preventing the formation of physiologically relevant interactions. Unsurprisingly, mutations of RNA binding proteins are common cause of various diseases (Gebauer et al, 2021).…”

Mechanism of an alternative splicing switch mediated by cell-specific and general splicing regulators

“…While many new RBPMS interactors were identified by affinity pulldown (Fig 6), ten proteins in our dataset are known RBPMS interactors including RBFOX2, MBNL1 and RBM14 (Huttlin et al, 2021, Lang et al, 2021, Rual et al, 2005, Yang et al, 2018. That most interactions are direct but enhanced by the presence of RNA (Fig.…”

“…While many new RBPMS interactors were identified by affinity pulldown (Fig 6), ten proteins in our dataset are known RBPMS interactors including RBFOX2, MBNL1 and RBM14 (Huttlin et al, 2021, Lang et al, 2021, Rual et al, 2005, Yang et al, 2018). That most interactions are direct but enhanced by the presence of RNA (Fig.6.C) is consistent with combinatorial models of splicing regulation, in which the low affinity of binary protein-protein interactions is tuned so as to enable specific cooperative assembly only upon regulated substrates with the correct combination of binding sites (Smith & Valcárcel, 2000).…”

RBPamp: Quantitative Modeling of Protein-RNA Interactionsin vitroPredictsin vivoBinding

“…For some RBPs (PUM1, RBM22, HNRNPA1), predicted affinity was not well correlated with CLIP signal, or differed between cell types, suggesting that additional factors may modulate the distribution of these RBPs in vivo. For example, joint binding of RNA by multiple RBPs, sometimes altering specificity, has been observed (Kuwasako et al, 2014; Leeper et al, 2010; Weidmann et al, 2016; Lang et al, 2021). Such joint binding could direct the protein to RNAs containing motifs distinct from those bound by the protein by itself in vitro, or could distort the shape of the relationship between affinity and eCLIP density.…”