Protocol to identify the RNA-binding domains of RBPs in a global scale

RBDmap employs UV crosslinking, oligo(dT) selection, partial proteolysis and quantitative proteomics to identify the protein regions engaged in RNA binding in a system-wide scale. Applied to HeLa cells it reported 1,174 RNA-binding sites mapping to 529 RBPs, many of which lacking known RNA-binding domains. A detail RBDmap protocol has now been released for the community in Nature protocols


The use of RBDmap can now be extended to other cell lines or organisms and can be used to profile in a global scale the behaviour of RNA-binding domains in response to different physiological conditions and stresses.”


Identification of RNA-binding domains of RNA-binding proteins in cultured cells on a system-wide scale with RBDmap. Castello A, Frese CK, Fischer B, Järvelin AI, Horos R, Alleaume AM, Foehr S, Curk T, Krijgsveld J, Hentze MW. Nat Protoc. 2017 Dec;12(12):2447-2464. doi: 10.1038/nprot.2017.106. Epub 2017 Nov 2. PMID:  29095441

The activity of TRIM25 is controlled by RNA

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.


Original publication:
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
BMC biology

Decapping host and viral RNAs

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.

 

 

Capture and proteomic analysis of single RNP species

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.

RNA2017 – Prague

We are back from the RNA2107 in Prague. Very exciting talks!

One highlight, Adrian Krainer talked about SPINRAZA, a new therapy against spinal muscular atrophy (SMA) based on antisense oligos. SMA produces the loss of motor neurons that ends up in muscle wasting and early dead (the life expectation of a new born baby with type 1 SMA is ten months). The recently developed treatment treatment has shown excellent results in phase III clinical trials. Adrian guided us through the conception and development of the drug and included emotive videos of how SPINRAZA improves the life of children affected with SMA. An inspiring talk that demonstrates that basic science is fundamental for advancing applied research.