A versatile assay for RNA-binding proteins in living cells

Abstract

RNA-binding proteins (RBPs) control RNA fate from synthesis to decay. Since their cellular expression levels frequently do not reflect their in vivo activity, methods are needed to assess the steady state RNA-binding activity of RBPs as well as their responses to stimuli. While electrophoresis mobility shift assays (EMSA) have been used for such determinations, their results serve at best as proxies for the RBP activities in living cells. Here, we describe a quantitative dual fluorescence method to analyze protein-mRNA interactions in vivo. Known or candidate RBPs are fused to fluorescent proteins (eGFP, YFP), expressed in cells, cross-linked in vivo to RNA by ultraviolet light irradiation, and immunoprecipitated, after lysis, with a single chain antibody fragment directed against eGFP (GFP-binding protein, GBP). Polyadenylated RNA-binding activity of fusion proteins is assessed by hybridization with an oligo(DT) probe coupled with a red fluorophore. Since UV light is directly applied to living cells, the assay can be used to monitor dynamic changes in RNA-binding activities in response to biological or pharmacological stimuli. Notably, immunoprecipitation and hybridization can also be performed with commercially available GBP-coupled 96-well plates (GFP-multiTrap), allowing highly parallel RNA-binding measurements in a single experiment. Therefore, this method creates the possibility to conduct in vivo high-throughput RNA-binding assays. We believe that this fast and simple radioactivity-free method will find many useful applications in RNA biology.

A short conserved motif in ALYREF directs cap- and EJC-dependent assembly of export complexes on spliced mRNAs

“…ALYREF contains different putative RNA-binding regions, of which RRM and RGG-rich regions of ALYREF were previously suggested as RNA-binding domains in living cells ( 58 ). However, we did not detect any interaction of the isolated RRM or RGG-rich regions with RNA.…”

Plant Desiccation Tolerance and its Regulation in the Foliage of Resurrection “Flowering-Plant” Species

“…A similar regulatory role to that of miRNA is exerted by RNA-binding proteins that modify mRNA activity and turnover of mRNA [214]. More than 1000 RNA-binding proteins have been identified from eukaryotes, but as yet none have been examined in relation to desiccation tolerance to the authors' knowledge.…”

Extensive cross-regulation of post-transcriptional regulatory networks in Drosophila

“…Several groups have used probes to purify specific target RNAs and then identify the associated proteins, though these approaches often require tagging the target RNA (for review, see McHugh et al 2014). Hentze (Strein et al 2014) and Parker (Mitchell et al 2013) have used oligo-dT to globally purify human and yeast cellular mRNA-protein complexes (mRNPs), respectively, and then identified the bound proteins but not the associated RNAs. However, very few studies have purified native RNP complexes and characterized both the RNA and protein components.…”