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Three hybrid

4 February 2011

 

Introduction.

The three hybrid approach allows the search of RNA interacting proteins, using the yeast Saccharomyces cerevisiae. We can either study the interaction between a specific prey protein and a bait RNA, or using a cDNA library to identify unknown RNA binding protein.

Principle.

As indicated by its name, the system is divided onto 3 parts:
- A fusion between the LEXA DNA binding domain and the MS2 protein. The latter specifically interacting with a RNA stem loop.
- A fusion between the MS2 stem-loop and the bait RNA (which can be either a stem-loop, a pseudoknot or an unstructured RNA sequence).
- The third part is a fusion between the transcription-activating domain of B42 protein and the prey protein (or library).

When the "?" protein interacts with the bait RNA the transcription activator is reconstituted, which induces the expression of the reporter genes (lacZ that gives a blue colour in presence of X-gal, and the HIS3 gene). These two reporter genes allow the selection of clones where an interaction occurs.

There are serious issues with this system, mainly due to many unspecific interactions between several proteins and the bait RNA or due to the activation of the transcription independently of the RNA (for example, a DNA binding protein that interacts directly with the promoter). However this is one of the very few systems testing the interaction between a protein and a RNA in vivo in an eukaryotic cell.

Issues

During a standard screening procedure there are a significant number of false positives. It is critical to analyse in detail each candidate sequence to be sure there is no direct interaction between this protein and the DNA promoter. It is important to test whether the interaction still occurs in presence of different bait RNA (if this is the case, the interaction is unspecific). It is worth to note that even when the interaction is specific this one can be irrelevant for the cell physiology. Indeed if the protein and the RNA are located in two separated cellular compartments, the physiological relevance is highly questionable.