Regulatory RNAs: charming gene management styles for synthetic biology applications

Abstract

RNAs have many important functional properties, including that they are independently controllable and highly tunable. As a result of these advantageous properties, their use in a myriad of sophisticated devices has been widely explored. Yet, the exploitation of RNAs for synthetic applications is highly dependent on the ability to characterize the many new molecules that continue to be discovered by large-scale sequencing and high-throughput screening techniques. In this review, we present an exhaustive survey of the most recent synthetic bacterial riboswitches and small RNAs while emphasizing their virtues in gene expression management. We also explore the use of these RNA components as building blocks in the RNA synthetic biology toolbox and discuss examples of synthetic RNA components used to rewire bacterial regulatory circuitry. We anticipate that this field will expand its catalog of smart devices by mimicking and manipulating natural RNA mechanisms and functions.

Keywords: RNA regulation; RNAs and biotechnology; riboswitches; small RNAs; synthetic RNAs; transcriptional control; translational control.

Figure 1. Universe of synthetic RNA devices and parts. An overview of the universe of RNA devices and parts that have been engineered in all three kingdoms of life. The red square delimits the space of RNA devices that this review focuses on. The blue arrow to the left of the picture represents how the complexity increases from bottom to top. The classification of RNA devices has been made consistent to previous work.

Figure 2. Gene expression control mechanisms of ligand-binding riboswitches. (A) Transcriptional elongation control by a ligand-activated riboswitch. In the absence of ligand, an anti-terminator stem is present allowing completion of transcription by a polymerase. A ligand binds a specific site in the aptamer and causes the formation of a terminator stem that arrests transcription and prevents the synthesis of a complete transcript (dashed line). The opposite mechanism in which transcription turns on upon binding has been identified in nature to a lesser extent (not shown in figure). (B) Translational initiation control by a ligand-activated riboswitch. In the presence of the ligand, a riboswitch undergoes a conformational change and sequesters the Ribosome-Binding Site (RBS) to prevent the ribosome from binding the mRNA. In the same way as with transcriptional controllers, there are examples of riboswitches that turn on translation upon binding (not shown in figure). (C) Translational control by an allosteric ribozyme. The glmS glucosamine-6-phosphate (GlcN6P)-activated ribozyme (aptazyme) is shown. In the absence of GlcN6P, translation is ON and the transcript is protected against degradation. In the presence of the ligand binding to the aptamer, the transcript is cleaved in the 5′ UTR by RNaseJ1 and is subject to decay.

Figure 3. Gene expression control mechanisms by bacterial sRNAs. (A) Transcription attenuation/enhancement. (A) sRNA binds to its target mRNA and causes a structural reconfiguration upon base-pairing, ultimately enhancing or attenuating transcription by the polymerase. (B) Translational control. Translational control is imparted by sRNAs in various ways: (1) A sRNA base-pairs to its target mRNA sequestering the Ribosome-Binding Site (RBS) and directly prevents translation initiation by the ribosomes. (2) A sRNA binds to the target mRNA at a distance from the RBS and the target mRNA suffers a structural change that indirectly affects ribosome binding. sRNA binding to its target can also enhance or inhibit mRNA decay by changing interactions with exonucleases and/or endonucleases.

Figure 4. Composability of riboswitches as a strategy for the synthesis of artificial RNA devices. Composability refers to the ability of a system to break down in units (parts) due to the system modularity and recombine in different configurations to satisfy specific human requirements. (A) Composability of riboswitches. Riboswitches can be decomposed and recombined for the synthesis of new devices with high modularity. An artificial riboswitch-based device is composed of a regulator (riboswitch), a signal (ligand), and an actuator (gene reporter). The regulator (inside the solid square) can be further decomposed into a sensor (aptamer) and an adaptor (expression platform that usually contains a terminator stem). (B) Composability of aptazymes (allosteric ribozymes). A synthetic catalytic device is composed of a catalytic regulator (aptazyme), a signal (ligand), and an actuator (gene reporter). The catalytic regulator (inside the solid box) can be further disassembled in a sensor (aptamer) and a scissile adaptor (ribozyme).

Figure 5. Composability of sRNAs as a strategy for the synthesis of artificial RNA devices. Composability, as for the case of riboswitches (Fig. 4) is the ability of a system to break down in units (parts) due to the system modularity and recombine in different configurations to satisfy specific human requirements. sRNAs are regulators of high modularity. An sRNA-based regulator can be broken down in two main parts: a sensor (target binding domain) and a stabilizer (that can include an Hfq-binding site and the transcriptional termination domain). In the context of a genetic device, the sRNA binds an mRNA target. In this case, the 5′ UTR of the target mRNA acts as an adaptor that transmits the signal to the gene reporter actuator. The combination of the sRNA and mRNA target comprises a functional synthetic device.

Figure 6. Dual and chimeric riboregulators. Based on the composability of these regulatory molecules, they can be recombined and assembled into a new regulator that exerts a completely different function. (A) Translational control by a small trans-acting RNA-responsive switch. Isaacs and collaborators designed a riboregulator combining a riboswitch-like sensor (YUNR motif), an adaptor that contains an anti-RBS sequence (crRNA) that can pair with the RBS and an sRNA-like effector molecule (taRNA). Translation is OFF (left) when the taRNA absent since crRNA sequesters the Ribosomal Binding Site (RBS). Translation turns ON (right) in the presence of the taRNA since it releases the RBS by recognizing the YUNR motif that leads to disruption of the CrRNA-RBS stem-loop. (B) Transcriptional control by a small trans-acting RNA-responsive switch. A transcriptional attenuator (from plasmid pT181) was engineered to be RNA sensitive. The sensor loop in the attenuator was evolved to detect a short antisense RNA (that acts as an effector molecule) via a kissing-loop interaction. This interaction exposes an intrinsic transcriptional terminator (the adaptor) to turn transcription OFF. (C) A ligand-responsive riboswitch activating an ncRNA. A chimeric regulator was engineered by using a natural aptamer as sensor and a non-coding antisense RNA (ncRNA) as the adaptor. By means of a kissing-loop interaction in the absence of the ligand, the aptamer interacts with the ncRNA hairpin inactivating its antisense function. In the presence of the ligand, the hairpin interaction is disrupted and the ncRNA recovers its regulatory functions. (D) An sRNA-responsive aptazyme. A trans-acting RNA-sensitive hallosteric ribozyme was engineered by designing a sensor (TR: trans-acting RNA responsive element) that detects the trans-acting RNA and coupling it with a Hammerhead ribozyme (HHR). In the absence of the trans-acting RNA (left), the aptazyme undergoes cleavage exposing the (initially occluded) ribosomal binding site and allowing translation. In contrast, upon binding of the trans-acting RNA to the TR element, the Hammerhead ribozyme undergoes a structural change that renders it catalytically inactive; this masks the RBS and prevents translation initiation.

Figure 7. Application of a translational regulator to construct complex genetic circuits. The following three examples use the crRNA-taRNA, a riboregulator that controls translation initiation (Fig. 5A, Isaacs et al., 2004), to engineer different synthetic systems. (A) An adaptor that converts regulators from translational to transcriptional. By fusing an adaptor to a translational regulator, the translational regulator becomes transcriptional regulator. The adaptor consists of a Ribosomal Binding Site (RBS), an Open Reading Frame (ORF) of a short leader peptide (tnaC), and a transcription termination region. By fusing this element to the translational regulator (e.g., crRNA-taRNA), translation of the adaptor is controllable (not shown). In the absence of the adaptor, regulation of GFP is at the translation initiation level by RBS sequestration. In contrast, by merging the adaptor, regulation of GFP is at the transcriptional elongation level; in this case, when the upstream translational regulator turns off synthesis of the adaptor, the transcription terminator in this element is exposed and arrests transcription of GFP (at the mRNA level). Likewise, when the translational regulator turns on synthesis of the adaptor, translation of GFP is activated at the protein level. (B) Riboregulated Transcription Cascade (RTC) with the ability to count. By arranging 3 genes controlled by the translational regulator (crRNA-taRNA) into a transcriptional cascade a genetic circuit is built with the ability to count. When P_BAD promoter is not induced, the crRNA element present in each gene blocks translation of the T7 and T3 RNA Polymerase (RNAP), and GFP. In contrast, when P_BAD is induced with arabinose, the level of fluorescence generated correlates with the number of pulses of arabinose fed into the system. (C) Switchboard for multi-sensing and metabolic pathway control. A series of rationally designed orthogonal variants of the crRNA-taRNA system (cr1, cr2…crn; taRNA1, taRNA2…taRNAn) are fused to multiple gene reporters (Reporter 1, Reporter 2…Reporter n) for simultaneous and independent regulation. The complex genetic circuit is arranged in parallel (each pair of crRNA-taRNA variants is under the control of a different promoter) to be able to sense multiple inputs and convert them into a measurable output (e.g., enzymatic and/or fluorescence reads).