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.
RBDmap was used to determine in a proteome-wide scale the protein regions engaged in RNA binding in living cells. It employs UV crosslinking, oligo(dT) capture and controlled proteolysis to isolate the protein regions interacting with polyadenylated RNA. Applied to HeLa cells RBDmap reported 1174 high probability RNA-binding sites in 529 proteins.
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.