A Fluorescent Complementation Assay for Investigating RNA-Protein Interactions in Vivo

A fluorescent complementation assay for investigating RNA-protein interactions in vivo

Grant Gelderman and Lydia Contreras                            

McKetta Department of Chemical Engineering, University of Texas at Austin, 200 E. Dean Keeton  St., Stop C0400, Austin TX 78712

Small non coding RNAs are known as powerful cellular regulators of translation, capable of promoting or inhibiting translation initiation by binding mRNAs changing their structure, promoting or catalyzing mRNA degradation, or by binding other regulatory proteins and affecting their activity [1]. The interactions of these RNAs and proteins are often part of a large regulatory pathway responsible for complex forms of genetic regulation. Many methods to study RNA-protein interactions are limited largely to in vitro studies such as, alanine scanning, mobility assays, SELEX, electron microscopy, X-ray diffraction, etc. While these methods are powerful analytical tools and are useful for determining and characterizing binding domains in the protein and RNA sequence, the findings of these studies do not always extrapolate to the interactions occurring in vivo. 

In this work, we present a method for studying RNA-protein interactions in vivo using fluorescent protein complementation [2] and flow cytometery. An advantage of this system is that it allows the interaction to occur in vivo and can elucidate how a complex system responds to stimuli from the environment by allowing single cell resolution to determine the average conditions of cell populations. We will demonstrate the proof of concept using a protein and an RNA from the carbon storage regulator (Csr) elements of E. coli (CsrA and CsrB, respectively). We evaluate the effect that mutations to CsrA and CsrB have on the fluorescent activity in order to probe the contribution of specific amino acid and nucleic acid residues in the RNA-protein interaction. Additionally, we present a rationale to screen for residues involved in RNA-protein interactions using random mutagenesis and flow cytometery. 

Our method for detecting and quantifying RNA-protein interactions can give useful insights into RNA-protein interactions in vivo. Additionally, this method can be used to provide a rationale for targeting specific RNA-protein regulatory elements and tuning their interaction in order to optimize cellular conditions for metabolic engineering.

                                  

[1]        Storz, G., Vogel, J. & Wassarman, K. Regulation by small RNAs in bacteria: expanding

frontiers. Molecular cell 43, 880–91 (2011).

[2]        Shyu, Y. & Hu, C.-D. Fluorescence complementation: an emerging tool for biological research. Trends in biotechnology 26, 622–30 (2008).