Riboswitches — To Kill or Save the Messenger
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《新英格兰医药杂志》
Humans are extraordinarily complex organisms; the intricacy of the underlying pattern of gene expression that orchestrates our development and governs our ability to respond to various stimuli is nothing short of marvelous. Every day of our lives, thousands upon thousands of genes are turned on or off, and the levels of messenger RNA (mRNA) expressed as a result of these genes increase or decrease in a coordinated and controlled manner. To orchestrate this complex series of molecular events, humans and other organisms have evolved sophisticated machinery that tightly regulates gene expression through a set of molecular switches — a challenge to the translational researcher who wishes to insert or manipulate a gene and control the resulting expression of a clinically important gene product for therapeutic purposes. A recent study by Yen and colleagues1 is therefore notable in its demonstration of a new, malleable method to control gene expression for therapeutic applications.
Taking cues from nature and RNA biochemists,2 Yen et al. exploited properties of RNA molecules to generate a new type of molecular switch that can be used to control the expression of a gene product of interest. The authors used a self-cleaving catalytic RNA as an mRNA self-destruction module (Figure 1). They showed that embedding this self-cleaving RNA motif in reporter genes effects the destruction of the motif-containing RNA — thus preempting translation of the RNA into protein.
Figure 1. The Use of a Riboswitch to Regulate Gene Expression.
A major obstacle to gene therapy is the difficulty of controlling the levels of gene expression in vivo. Yen and coworkers1 recently described a new approach. They embedded an RNA self-destruction motif in the noncoding region of a reporter gene. After transcription, an mRNA containing the motif cleaves itself and undergoes degradation, since the riboswitch is in the destruction mode. To turn the riboswitch to the salvage mode, a regulator is added that binds and inactivates the self-destruction motif, allowing the mRNA to survive and be translated into protein.
To convert this self-destruction system into a regulatable "riboswitch," the authors then exploited the ease with which one can alter the structure and activity of RNA using small molecules or short oligonucleotides that bind to specific target RNAs.2,3,4,5 Yen et al. used both classes of agent to target the RNA self-destruction motif itself. The binding of such a modulatory agent to the riboswitch prevents the riboswitch from destroying itself and allows the translation of the RNA to produce the protein of interest (Figure 1).
Using this destroy-the-messenger approach as opposed to a save-the-messenger approach, the authors showed that by turning the riboswitch from the destruction mode to the salvage mode, gene expression can be changed from very low levels to relatively high levels (an increase by a factor of up to 2000) in mammalian cells grown in culture. Reassuringly, they also showed that the method works in living animals. The injection of a reporter gene that contained a riboswitch into the eyes of mice resulted in moderate expression of the reporter gene product; subsequent injection of a small-molecule regulator increased the expression of the gene product by a factor of up to 190 in the eyes of the treated animals.
Yen et al. went on to show that riboswitches may work when inserted into various other types of mRNA. Thus, riboswitches can, in principle, be used to control the expression of a variety of gene products. Moreover, it is likely that they can be designed to respond to a variety of different modulatory agents.2,3
So how might riboswitches be used in the clinic? In the setting of gene therapy, the expression of therapeutic proteins must be tightly and properly controlled for most gene therapies to be efficacious and for many of them to be safe in complex organisms such as humans. For example, for the genetic treatment of type 1 diabetes, the ability of transferred genes to modulate insulin production by means of riboswitches that sense and respond to glucose levels should not only improve the efficacy of insulin therapy but also curb the side effects that would be expected to result from the inappropriate expression of insulin.
However, as with the development of any new therapy, a number of major issues must be addressed before riboswitches can be evaluated in the clinical setting. First, riboswitches that respond to clinically relevant drugs or metabolites must be developed and shown to function when they are incorporated into clinically relevant genes. Next, riboswitches must be tested in a variety of animal tissues. And finally, as with all gene-therapy strategies, methods for the efficient and safe delivery of riboswitches into endogenous genes or as part of exogenously administered therapeutic genes must be developed and optimized. Future studies that address these issues will ultimately reveal whether riboswitches can fulfill their clinical potential.
Source Information
From the Department of Surgery, Duke University Medical Center, Durham, N.C.
References
Yen L, Svendsen J, Lee JS, et al. Exogenous control of mammalian gene expression through modulation of RNA self-cleavage. Nature 2004;431:471-476.
Mandal M, Breaker RR. Gene regulation by riboswitches. Nat Rev Mol Cell Biol 2004;5:451-463.
Breaker RR. Engineered allosteric ribozymes as biosensor components. Curr Opin Biotechnol 2002;13:31-39.
Werstuck G, Green MR. Controlling gene expression in living cells through small molecule-RNA interactions. Science 1998;282:296-298.
Rusconi CP, Scardino E, Layzer J, et al. RNA aptamers as reversible antagonists of coagulation factor IXa. Nature 2002;419:90-94.(Bruce A. Sullenger, Ph.D.)
Taking cues from nature and RNA biochemists,2 Yen et al. exploited properties of RNA molecules to generate a new type of molecular switch that can be used to control the expression of a gene product of interest. The authors used a self-cleaving catalytic RNA as an mRNA self-destruction module (Figure 1). They showed that embedding this self-cleaving RNA motif in reporter genes effects the destruction of the motif-containing RNA — thus preempting translation of the RNA into protein.
Figure 1. The Use of a Riboswitch to Regulate Gene Expression.
A major obstacle to gene therapy is the difficulty of controlling the levels of gene expression in vivo. Yen and coworkers1 recently described a new approach. They embedded an RNA self-destruction motif in the noncoding region of a reporter gene. After transcription, an mRNA containing the motif cleaves itself and undergoes degradation, since the riboswitch is in the destruction mode. To turn the riboswitch to the salvage mode, a regulator is added that binds and inactivates the self-destruction motif, allowing the mRNA to survive and be translated into protein.
To convert this self-destruction system into a regulatable "riboswitch," the authors then exploited the ease with which one can alter the structure and activity of RNA using small molecules or short oligonucleotides that bind to specific target RNAs.2,3,4,5 Yen et al. used both classes of agent to target the RNA self-destruction motif itself. The binding of such a modulatory agent to the riboswitch prevents the riboswitch from destroying itself and allows the translation of the RNA to produce the protein of interest (Figure 1).
Using this destroy-the-messenger approach as opposed to a save-the-messenger approach, the authors showed that by turning the riboswitch from the destruction mode to the salvage mode, gene expression can be changed from very low levels to relatively high levels (an increase by a factor of up to 2000) in mammalian cells grown in culture. Reassuringly, they also showed that the method works in living animals. The injection of a reporter gene that contained a riboswitch into the eyes of mice resulted in moderate expression of the reporter gene product; subsequent injection of a small-molecule regulator increased the expression of the gene product by a factor of up to 190 in the eyes of the treated animals.
Yen et al. went on to show that riboswitches may work when inserted into various other types of mRNA. Thus, riboswitches can, in principle, be used to control the expression of a variety of gene products. Moreover, it is likely that they can be designed to respond to a variety of different modulatory agents.2,3
So how might riboswitches be used in the clinic? In the setting of gene therapy, the expression of therapeutic proteins must be tightly and properly controlled for most gene therapies to be efficacious and for many of them to be safe in complex organisms such as humans. For example, for the genetic treatment of type 1 diabetes, the ability of transferred genes to modulate insulin production by means of riboswitches that sense and respond to glucose levels should not only improve the efficacy of insulin therapy but also curb the side effects that would be expected to result from the inappropriate expression of insulin.
However, as with the development of any new therapy, a number of major issues must be addressed before riboswitches can be evaluated in the clinical setting. First, riboswitches that respond to clinically relevant drugs or metabolites must be developed and shown to function when they are incorporated into clinically relevant genes. Next, riboswitches must be tested in a variety of animal tissues. And finally, as with all gene-therapy strategies, methods for the efficient and safe delivery of riboswitches into endogenous genes or as part of exogenously administered therapeutic genes must be developed and optimized. Future studies that address these issues will ultimately reveal whether riboswitches can fulfill their clinical potential.
Source Information
From the Department of Surgery, Duke University Medical Center, Durham, N.C.
References
Yen L, Svendsen J, Lee JS, et al. Exogenous control of mammalian gene expression through modulation of RNA self-cleavage. Nature 2004;431:471-476.
Mandal M, Breaker RR. Gene regulation by riboswitches. Nat Rev Mol Cell Biol 2004;5:451-463.
Breaker RR. Engineered allosteric ribozymes as biosensor components. Curr Opin Biotechnol 2002;13:31-39.
Werstuck G, Green MR. Controlling gene expression in living cells through small molecule-RNA interactions. Science 1998;282:296-298.
Rusconi CP, Scardino E, Layzer J, et al. RNA aptamers as reversible antagonists of coagulation factor IXa. Nature 2002;419:90-94.(Bruce A. Sullenger, Ph.D.)