J. K. Wickiser Lab

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Reprogramming cellular behavior with RNA controllers responsive to endogenous proteins.

Here are two reviews of a cool paper detailing synthetic RNA switches in the cell.

“I found this paper interesting because the authors describe an innovative step towards engineering a regulatory system that can change the expression of targeted genes.

More importantly, their system senses specific endogenous signals defined by users and responds by directing the cells to an alternative behavior. Indeed, they showed that endogenous signalling pathways can be rerouted to induce novel cellular phenotypes, including apoptosis. Their sensing device is an RNA aptamer that, upon binding to a specific protein, changes its structure and function to modulate the alternative spicing of the gene in which it has been inserted. Their work emphasizes the efficacy and versatility of RNA aptamers for engineering gene regulation systems and hence for gene therapy.
Competing interests: None declared”

Cite this evaluation
DesGroseillers L: “I found this paper interesting because the authors describe an innovative step towards engineering a…” Evaluation of: [Culler SJ et al. Reprogramming cellular behavior with RNA controllers responsive to endogenous proteins. Science. 2010 Nov 26; 330(6008):1251-5; doi: 10.1126/science.1192128]. Faculty of 1000, 03 Feb 2011. F1000.com/7626956

“Molecular biologists usually alter cellular functions by overexpressing, mutating, or knocking down components of transcription factor and signal transduction networks. However, a method for reprogramming cellular behavior in response to any user-defined component of endogenous signaling networks would provide an important tool both for measuring the activities of these networks and using them to tailor cellular function. Culler et al. have managed to devise such a method by engineering what they call, “RNA-based sensing actuation devices”.

Basically, these devices comprise a three-exon mini-gene with two introns. The middle exon includes a translational stop codon that prevents translation of the open reading frame (ORF) encoded in the 3’-most exon. The critical component in this system is an RNA aptamer incorporated into the intronic sequences flanking the middle exon. The aptamer is first generated by in vitro selection to bind to a specific protein ligand. The authors show that association of the specific ligand with the inserted aptamer can act to alternatively splice the mini-gene such that the middle exon containing the stop codon is either included or excluded. The mechanism through which aptamer-ligand interaction influences splicing is unclear and the exact position of the aptamer within the intronic sequences required to trigger alternative splicing must be determined empirically for each aptamer. In principle, these devices could be employed to sense any endogenous protein and to respond by expressing any protein encoded in the 3’ exon ORF.

The authors provide several compelling demonstrations that their sensing devices work quite well. For example, inclusion of an aptamer that binds phage MS2 coat protein results in expression of the green florescent protein (GFP) signal encoded in the 3’ exon ORF in response to expression of the coat protein. Moreover, treatment of cells with exogenous factors that stimulate endogenous nuclear factor kappa B (NFkB)- or Wnt-signaling pathways also produce a robust GFP signal from mini-genes with aptamers binding NFkB p65 or beta-catenin, respectively. They also show that insertion of two aptamers can increase the response and that, at least in some cases, multiple ligands act to evoke a synergistic response. Lastly, they demonstrate that the system can be employed to regulate cell fate decisions. Thus, a mini-gene designed to ‘sense’ beta-catenin and produce the herpes simplex virus thymidine kinase from the 3’ ORF will trigger cell death in the presence of Wnt ligand and the pro-drug Ganciclovir.

This work underscores the versatility of RNA as both a biological sensor and regulator and suggests that the engineering of synthetic networks is within reach (also see related perspective by Liu and Arkin {1}).

References:
{1} Liu and Arkin, Science 2010, 330:1185-6 [PMID:21109657].
Competing interests: None declared”

Cite this evaluation
Eisenman R: “Molecular biologists usually alter cellular functions by overexpressing, mutating, or knocking down components of transcription…” Evaluation of: [Culler SJ et al. Reprogramming cellular behavior with RNA controllers responsive to endogenous proteins. Science. 2010 Nov 26; 330(6008):1251-5; doi: 10.1126/science.1192128]. Faculty of 1000, 19 Jan 2011. F1000.com/7626956

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This entry was posted on Wednesday, August 24, 2011 at 5:26 am and is filed under Current Data Papers. You can follow any responses to this entry through the RSS 2.0 feed. You can leave a response, or trackback from your own site.

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