Cells are built up of complex molecular networks that control the cell's responses to the environment and cell-fate decisions such as proliferation, differentiation or apoptosis. These molecular networks process information and link it to new cellular behaviors. To study and manipulate the wiring that underlies cellular function, one needs tools that allow interfering with these signaling networks in a precise and controlled manner.

Several different bioengineering strategies now allow researchers to do this. These perturbative tools are normally composed of a 'sensing' module that responds to particular stimuli and an 'effector' module that alters cellular behavior by affecting processes such as gene expression, enzymatic activity or protein translocation.

Christina Smolke and her colleagues at Stanford University are interested in using RNA for building such tools. In new work, her group has engineered 'RNA controllers', molecules that respond to specific cellular proteins by modulating the expression of other proteins. “We took aptamers—which are basically RNA molecules that will bind to specific proteins—and used them as our sensing components and then linked them to an RNA-based gene regulatory system based on alternative splicing,” she explains.

In these RNA devices, a gene of interest is placed downstream of a minigene composed of three coding regions (exons) separated by two noncoding regions (introns) that contain the aptamer. The middle exon has a stop codon such that translation of the gene of interest is high when the exon is excluded. The binding of a protein to the aptamer affects the splicing pattern of this RNA molecule through mechanisms that are to be determined, resulting in changes in expression of the gene of interest. The system is highly flexible: depending on where the aptamer sequence is placed in the intron, its binding to the protein can affect splicing differently, increasing or decreasing the final levels of the gene product. “You can also swap in different sensors [aptamers] that respond to different proteins in the cell and maintain the same sort of functional genetic device,” says Smolke, or build devices that respond to multiple protein inputs by incorporating several aptamers, as is shown in this work.

RNA controllers that respond to both endogenous and heterologous nuclear proteins can be built in this way. Graduate student Stephanie Culler and postdoc Kevin Hoff in Smolke's laboratory engineered RNA devices that respond to proteins involved in the NF-κB and Wnt signaling pathways showing, in human cultured cells, that they could rewire these pathways and produce new cellular behaviors. By placing a suicide gene as the effector module of β-catenin and NF-κB responsive devices, the group could trigger targeted cell death upon stimulation of these pathways.

Mechanism of action of RNA controllers. Binding of the protein input to the sensor alters the splicing pattern of the RNA molecule by enhancing or suppressing alternative exon exclusion, resulting in different production levels of the gene of interest, X.

Smolke is interested in integrating RNA controllers in different biological systems. By looking at how these devices can be used to control signaling pathways that affect the cell cycle, for example, her group is looking ahead to their future application for disease treatment and stem-cell engineering.

These RNA-based devices are not only one of the first ones demonstrated to respond to native proteins but also, because they are based on the use of alternative splicing, they offer endless possibilities for gene-expression regulation. One could imagine future developments of more complex types of controllers based on similar strategies as the one used by the Smolke group. For example, by regulating the inclusion of exons that actually encode for functional protein domains, one could control what splicing variant of a protein is produced or where it is localized in the cell. Bioengineering and synthetic biology are rapidly growing fields and their application to biological studies will enable fine control of cellular function and also aid in further refining these promising techniques.