Recent publications from the lab
Specific RNP capture with antisense LNA/DNA mixmers. Rogell BM, Fischer B, Rettel M, Krijgsveld J, Castello A, Hentze MW. RNA. 2017 May 5. pii: rna.060798.117. doi: 10.1261/rna.060798.117. PMID: 28476952
Plant RNA Interactome Capture: Revealing the Plant RBPome. Bach-Pages M, Castello A, Preston GM. Trends Plant Sci. 2017 May 3. pii: S1360-1385(17)30071-7. doi: 10.1016/j.tplants.2017.04.006. PMID: 28478905
Comprehensive Identification of RNA-Binding Domains in Human Cells. Castello A, Fischer B, Frese CK, Horos R, Alleaume AM, Foehr S, Curk T, Krijgsveld J, Hentze MW. Mol Cell. 2016 Aug 18;63(4):696-710. doi: 10.1016/j.molcel.2016.06.029. Epub 2016 Jul 21.
The Cardiomyocyte RNA-Binding Proteome: Links to Intermediary Metabolism and Heart Disease. Liao Y, Castello A, Fischer B, Leicht S, Föehr S, Frese CK, Ragan C, Kurscheid S, Pagler E, Yang H, Krijgsveld J, Hentze MW, Preiss T. Cell Rep. 2016 Aug 2;16(5):1456-69. doi: 10.1016/j.celrep.2016.06.084. Epub 2016 Jul 21. PMID: 27452465
Global changes of the RNA-bound proteome during the maternal-to-zygotic transition in Drosophila. Sysoev VO, Fischer B, Frese CK, Gupta I, Krijgsveld J, Hentze MW, Castello A, Ephrussi A. Nat Commun. 2016 Jul 5;7:12128. doi: 10.1038/ncomms12128. PMID: 27378189
The new (dis)order in RNA regulation. Järvelin AI, Noerenberg M, Davis I, Castello A. Cell Commun Signal. 2016 Apr 6;14:9. doi: 10.1186/s12964-016-0132-3. Review. PMID: 27048167
Identification of RNA-binding Proteins in Macrophages by Interactome Capture. Liepelt A, Naarmann-de Vries IS, Simons N, Eichelbaum K, Föhr S, Archer SK, Castello A, Usadel B, Krijgsveld J, Preiss T, Marx G, Hentze MW, Ostareck DH, Ostareck-Lederer A. Mol Cell Proteomics. 2016 Aug;15(8):2699-714. doi: 10.1074/mcp.M115.056564. PMID: 27281784
Datasets – the emerging universe of RBPs
The RNA interactome dataset (Castello et al., Cell, 2012) compiles the repertoire of RNA-binding proteins (RBPs) identified by RNA interactome capture in HeLa cells. The protein maps is linked to the information available on these proteins, including their domain architecture and the presence of disordered repetitive motifs.
The RBDmap dataset depicts the distribution of RNA-binding sites within the RBPs identified by RNA interactome capture. The RBDmap protocol combines controlled proteolysis with ultraviolet crosslinking and oligo(dT) capture to narrow down the protein regions engaged in RNA binding (Castello et al., Mol Cell 2016). This method was also applied to murine cardiomyocytes.
In Castello et al., Trends in Genetics 2013, we generated a RBPs and disease that compilates the disease-associated mutations linked to known and newly identified RBPs. We noticed that protein disordered regions are enrichment in these Mendelian mutations.
Protocols to characterise RBPs
RNA interactome capture was developed to identify the repertoire of RNA-binding proteins (RBPs) of living cells. Applied to HeLa cells, it revealed 860 high confidence RBPs, many of which were previously unknown to bind RNA. RNA interactome capture has been adapted to primary cells, uni and pluricellular organisms, including plants, and to study subcellular organelles.It has also been applied to study the dynamics of the RNA-binding proteome during maternal-to-zygotic transition in drosophila embryos.
We recently published a new method to capture Specific RNP capture and their RNA-bound proteome in cell-free systems or culture cells. This protocol, termed Specific RNP capture, uses UV crosslinking and lock nucleic acid (LNA) probes. It was used to identify the RNA-bound proteome of a reporter transcript containing the sex-lethal (sxl) RNA-binding sites and the 18S and 28S rRNA.
The dual fluoresence RNA-binding assay is used to measure the RNA-binding activity of a giving protein in cultured cells or organisms. In brief, the known or putative RBP is expressed as a fusion to eGFP and immunoprecipitated with the GFP_Trap_A single chain Lama alpaca-derived nanobody. The presence of RNA is revealed by hybridisation with oligo(dT) fused to a red or far red fluorophore. Thus, the red to green fluorescence ratio serves as a proxy or poly(A) RNA molecule per protein. This method can be adapted to mid-throughput experiments using the GFP_Multitrap, a GFP_Trap coated 96 well plate.
We are happy to provide more details of these protocols as well as advise on how to implement them.
Funds supporting our work
We thank the following funders for supporting our work.
The MRC career development award provides up to five years’ support for outstanding post-doctoral researchers who wish to consolidate their research skills and make the transition from post-doctoral research trainee to independent investigator. We thank the MRC for its support.
The Early Career Researcher John Fell funds is seedcorn and start-up grant that provides staff and funds to stimulate applications to external agencies.
The CRUCK Oxford Centre developmental funds provide pum-priming funds for proof-of-princinple projects.
Manuel Garcia Moreno is funded by the Marie Skłodowska-Curie postdoctoral fellowship.