Abstract
RNA-binding ubiquitin ligases (RBULs) have the potential to link RNA-mediated mechanisms to protein ubiquitylation. Despite this, the cellular functions, substrates and interaction partners of most RBULs remain poorly characterized. Affinity purification (AP) combined with quantitative mass spectrometry (MS)-based proteomics is a powerful approach for analyzing protein functions. Mapping the physiological interaction partners of RNA-binding proteins has been hampered by their intrinsic properties, in particular the existence of low-complexity regions, which are prone to engage in non-physiological interactions. Here, we used an adapted AP approach to identify the interaction partners of human RBULs harboring different RNA-binding domains. To increase the likelihood of recovering physiological interactions, we combined control and bait-expressing cells prior to lysis. In this setup, only stable interactions that were originally present in the cell will be identified. We exploit gene function similarity between the bait proteins and their interactors to benchmark our approach in its ability to recover physiological interactions. We reveal that RBULs engage in stable interactions with RNA-binding proteins involved in different steps of RNA metabolism as well as with components of the ubiquitin conjugation machinery and ubiquitin-binding proteins. Our results thus demonstrate their capacity to link posttranscriptional regulation with the ubiquitin system.
Highlights
Posttranscriptional RNA processing provides a fundamental step in the regulation of gene expression
We analyzed the interaction profiles of six human RNA-binding ubiquitin ligases (RBULs) containing different RNA-binding domains using a modified affinity purification (AP) approach coupled to LC-mass spectrometry (MS)/MS
Previous studies have suggested that RNA-binding proteins (RBPs) are prone to engage in non-physiological interactions that hamper the identification of physiological interaction partners[7,8]
Summary
Posttranscriptional RNA processing provides a fundamental step in the regulation of gene expression. Recent large-scale mRNA interaction profiling studies have demonstrated that eukaryotic cells express >1,200 RBPs1–3 In addition to their RNA binding capability, many of these RBPs possess catalytic activities, introducing an additional layer of complexity into posttranscriptional regulation[1,3]. A bait protein and its interaction partners are purified by antibodies or affinity resins, and the interaction partners are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) This approach allows to efficiently discriminate physiological interactions from background binders, when combined with quantitative MS approaches such as stable isotope labeling with amino acids in cell culture (SILAC)[5].
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
