The E3 ubiquitin ligase TRIM25 is an antiviral factor recently discovered to bind RNA by the RNA interactome studies (Castello et al., 2012 and Kwon et al., 2013). In a recent work led by our collaborator Gracjan Michlewsky (Wellcome Centre for Cell Biology, University of Edinburgh), we dissected how this protein binds to RNA and what are the consequences of this interaction in TRIM25 function. We discovered that TRIM25 binds RNA via its PRY/SPRY domain and that the interaction with RNA enhances TRIM25 E3 ligase activity, which is necessary for its antiviral role. Using CLIP analyses we showed that TRIM25 binds G-rich sequences present in hundreds of cellular RNAs. Moreover, We discovered that TRIM25 controls the levels of a key component in the interferon response pathway, ZAP (also known as PARP13 and ZC3HAV1).
In conclusion, the E3 ligase activity of TRIM25 is controlled by RNA, breaking once more the view that proteins act on RNA and not the opposite.
RNA-binding activity of TRIM25 is mediated by its PRY/SPRY domain and is required for ubiquitination
Nila Roy Choudhury, Gregory Heikel, Maryia Trubitsyna, Peter Kubik, Jakub S. Nowak, Shaun Webb, Sander Granneman, Christos Spanos, Juri Rappsilber, Alfredo Castello and Gracjan Michlewski
In a recent work with Yolanda Revilla’s lab (CBMSO, Madrid) published in the Journal of Virology, we investigated the role in RNA metabolism of a protein from a complex DNA virus, called African swine fever virus (ASFV). This protein exhibits high homology with cellular decapping enzymes and thus can potentially remove the cap structure from the RNA body triggering degradation. We show that this protein interacts with viral and cellular mRNAs in infected cells. This interaction results in decreased levels of both types of transcripts, agreeing with a putative role as virus-encoded decapping activity. We propose that the degradation of RNA triggered by this protein is key to control gene expression in ASFV infected cells.
It is challenging to determine the composition of a given ribonucleoprotein. We recently approached this problem by adapting the original RNA interactome capture protocol (Castello et al., Cell 2012), to the use of specific antisense LNA probes to capture specific RNA species. We use this method to elucidate the composition of luciferase containing reporters and ribosomal RNA in vitro and in vivo. We were able to recapitulate well-established protein-RNA interactions and to discover new ones.
Specific RNP capture with antisense LNA/DNA mixmers. Rogell B, Fischer B, Rettel M, Krijgsveld J, Castello A, Hentze MW. RNA. 2017 Aug;23(8):1290-1302. doi: 10.1261/rna.060798.117. Epub 2017 May 5.
A new method, built on RNA interactome capture, reports a comprehensive atlas of RNA-binding domains. This mass spectrometry based approach, referred to as RBDmap, makes use of UV crosslinking, oligo(dT) capture, partial proteolysis and mass spectrometry to identify the protein regions in close contact with RNA. Applied to HeLa and HL-1 cardiomyocytes, this method revealed more than thousand RNA-binding sites in hundreds of RBPs. This sites map not only to classical RNA-binding domains, but also to proteins lacking known RNA-binding architectures. RNA-binding sites overlap with protein-protein interaction domains, enzymatic cores and disordered regions. These sites are enriched in known post-translational modifications and disease-associated mutations and thus the functional implications of these novel RNA-binding domains deserve consideration.
Comprehensive Identification of RNA-Binding Domains in Human Cells. Alfredo Castello, Bernd Fischer, Christian K. Frese, Rastislav Horos, Anne-Marie Alleaume, Sophia Foehr, Tomaz Curk, Jeroen Krijgsveld, Matthias W. Hentze. Mol Cell. DOI: http://dx.doi.org/10.1016/j.molcel.2016.06.029
The Cardiomyocyte RNA-Binding Proteome: Links to Intermediary Metabolism and Heart Disease. Yalin Liao, Alfredo Castello, Bernd Fischer, Stefan Leicht, Sophia Föehr, Christian K. Frese, Chikako Ragan, Sebastian Kurscheid, Eloisa Pagler, Hao Yang, Jeroen Krijgsveld5, Matthias W. Hentze, Thomas Preiss. Cell Reports. DOI: http://dx.doi.org/10.1016/j.celrep.2016.06.084
The current paradigm of RNA-binding proteins is that they contain regions, or domains, that fold tightly into an ordered interaction platform that mediate RNA binding. In this review, we describe how this paradigm has been challenged by studies showing that other, hitherto neglected regions in RNA-binding proteins, which in spite of being intrinsically disordered, can play key functional roles in protein-RNA interactions. Proteins harbouring such disordered regions are involved in virtually every step of RNA regulation and, in some instances, have been implicated in disease. Based on exciting recent discoveries that indicate their unexpectedly pervasive role in RNA binding, we propose that the systematic study of disordered regions within RNA-binding proteins will shed light on poorly understood aspects of RNA biology and their implications in health and disease.
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