(15c) 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).