An organism's genome encodes all of the biochemical instructions needed to produce a living cell.  The transcriptional programs of a cell, however, are dynamic, changing as the cell develops, grows and responds to alterations in the environment.  The control of gene expression is not well understood but all living cells require it.  Understanding these networks is fundamental for understanding human health and combating disease.  Because intrinsic properties of RNA make them ideal for rapid switches that modulate gene expression in response to changes in environment, the Liu Lab hypothesizes that novel, functional RNA motifs represent ubiquitous elements in numerous gene networks that allow cells to develop, grow, adapt and survive.  The broad goals of our research are to identify and understand, at a molecular level, the many ways by which RNA contributes to normal cell function, and to apply this gathered knowledge to the development of novel tools and potential therapeutics. 

We will use a model organism to study the control of gene expression by non-canonical RNA: the prokaryotic Vibrio cholerae.  The following are two on-going projects in the lab that address our interests and goals:

• Investigate the mechanisms by which non-canonical RNAs mediate bacterial survival.
  
  
• Engineer biosensors to monitor pathogenesis regulation in V. cholerae.

V. cholerae is a model organism exceptionally well suited for the study of non-canonical, regulatory RNA.  The causative agent of cholera, V. cholerae is an enteric pathogen with a similar genome size and gene content as pathogenic Escherichia coli and Salmonella spp.  These latter bacteria are responsible for many human food-borne illness outbreaks in the United States each year, and all three represent health-care challenges in developing nations (www.cdc.gov).  V. cholerae survive in both animal hosts and aquatic ecosystems, providing models for both human and environmental pathogens (Figure 1).  These bacteria must have evolved sophisticated signal transduction systems to survive in these diverse niches, providing ample opportunity to investigate gene networks for regulatory RNAs.

Figure 1. The life cycle of V. cholerae alternates between aquatic resevoirs such as ponds or estuaries, and the human small intestine.

During my post-doctoral studies, I hypothesized that unidentified small non-coding RNAs (sRNAs) play integral roles in the regulation of genes needed for environmental adaptation by V. cholerae.  To directly address this hypothesis, I developed a new method, sRNA-Seq, to investigate the entire repertoire of sRNAs in any given organism.  This method combines well-established direct cloning protocols and the power of deep sequencing technology.  I used this protocol and bioinformatic analysis to investigate the sRNA component of the V. cholerae transcriptome.  By using sRNA-Seq, we were able to identify 500 new intergenic sRNAs and 127 antisense sRNAs in a limited number of growth conditions examined.
 
Most characterized sRNAs elicit their regulatory effect by base-pairing with a target mRNA, and either destabilize the mRNA or affect its translation (Figure 2).  The Liu Lab proposes to use biochemical and molecular biology techniques to investigate these newly identified V. cholerae sRNAs.  The Liu Lab will initially focus on the characterization of sRNAs involved in the regulation of carbon metabolism.  The ability to take up and process different carbohydrates allow bacteria to adapt to multiple environments and growth conditions; we have identified sRNAs that may play an important role in gene regulatory pathways involved in such adaptation.  We will use biochemical and mutagenesis studies to reveal the functions and targets of these sRNAs.  We will also investigate structure/function relationships of sRNA sequences.  This work will significantly contribute to our understanding of bacterial survival mechanisms, potentially leading to new insights towards combating pathogens.

Figure 2. Mechanisms by which sRNAs can affect gene expression in bacteria.

The Liu Group is also very interested in projects that involve the utility of RNA as a tractable therapeutic and diagnostic tool.  Presently, there are a half-dozen pharmaceutical companies that have active programs investigating RNA-based therapeutics.  An RNA aptamer used to treat clot formation has recently completed Phase I clinical trials.  In addition, advances in transcriptional profiling, the measurement of total RNA populations in a cell, are paving the way towards personalized medicine in the clinic.  Several labs have also considered the utility of non-canonical RNA as a research tool.

Towards this end, we hypothesize that a bioengineered RNA can be used to investigate the regulation of pathogenesis in V. cholerae.  From bacteria to humans, complex signaling networks are used to detect extracellular signals (first messengers) that are then channeled through intracellular pathways, resulting in specific biochemical changes that allow bacteria to adapt to changes in environment.  Cytoplasmic small molecules, "second messengers", are also involved in these intracellular pathways.  For example, bacteria use the molecule cAMP to signal changes in glucose concentrations outside the cell.  In a similar vein, humans use hormones to direct specific biochemical responses from cells.  One small molecule, cyclic diguanylate (c-di-GMP), is an important second messenger in many pathogens including V. cholerae.

Previous work suggests that in V. cholerae c-di-GMP mediates the transition of the bacteria from a virulent lifestyle inside the human host to a biofilm-associated lifestyle in aquatic reservoirs.  These previous studies, however, depended primarily on forced changes in c-di-GMP levels.  The natural fluctuations of intracellular c-di-GMP concentrations throughout the V. cholerae life cycle remain uncharacterized.  We hypothesize that monitoring c-di-GMP levels in wild-type V. cholerae will significantly contribute to our understanding of the role of this second messenger in regulating pathogenesis.  We propose using directed evolution techniques to engineer biosensors that will rapidly signal changes in intracellular c-di-GMP concentrations as V. cholerae transitions through its life cycle.  Directed evolution is a method that uses the principles of natural selection to generate (evolve) proteins, DNA, or RNA with new, desirable properties.