Riboswitches are at the heart of many RNA-based technologies and they are the link between SELEX-based aptamer development and applications inside the cell. Today, three Fellows of our network answer questions about riboswitches: Sara tells us where they came from, Adrien explains how we can use them, and Eduardo forecasts the future of riboswitch design.
Why did riboswitches develop? How do they fit into the story of evolution?
SARA: “Adaptation to surroundings is essential for all living organisms, from the smallest bacteria to us. There are many ways a cell can sense the environment, most of which include complex pathways that coordinate like the best orchestra to allow accurate and efficient responses.
“The term “RNA world” refers to the hypothesis that self-replicating ribonucleic acid molecules (RNA) are the precursors of all current life forms on Earth. Imagine, for a moment, a living cell with no proteins, no lipids…and no DNA (deoxyribonucleic acid). How could it sense the environment and, more importantly, adapt to it? The answer may lie in the RNA molecule.
“There are special RNA molecules able to act as direct sensors of both external and internal changes, with no need of proteins or cofactors! These molecules, so-called riboswitches (RIBOnucleic acid SWITCH) are present in the vast majority of bacteria species where they serve as sensors for vast range of ligands (amino acids, anions, metals, purines, cofactors…).
“Some riboswitch classes, particularly those that sense molecules like TPP (thiamine pyrophosphate; a eukaryote-specific riboswitch), AdoCbl (adenosylcobalamin), and FMN (flavin mononucleotide) are exceptionally widespread (see figure, Barrick and Breaker 2007). These riboswitch ligands are thought to be molecular relics from an RNA World. Furthermore, the most widespread riboswitch classes have some of the most complex aptamer structures. These information-rich structures are unlikely to have emerged during late evolution. Rather, widespread complex riboswitches most likely have an early and perhaps RNA World origin.
“The main function of riboswitches is to modulate gene expression (and thereby adapt to environmental changes) by ligand-induced conformational changes using a wide range of means, in many cases organism-specific and even species-specific.“
How can the complexity and variation of natural riboswitches be used in synthetic systems?
ADRIEN: “Based on the principles of riboswitch regulation, a versatile set of riboswitches have been engineered. By combining RNA-based sensing domains with regulatory domains, a riboswitches can serve as a synthetic RNA regulator with multiple properties. Many tools have been developed exploiting these riboswitch properties for systems with conditional gene expression.
“Most mechanisms of engineered riboswitches used in bacteria involve either the Shine-Dalgarno sequence (a conserved nucleotide sequence where the ribosome can bind and produce the protein encoded in the gene upstream), or the terminator (a sequence which have a specific folding pattern which physically stops the RNA polymerase).
(A), example of regulation of gene expression with the Shine-Dalgarno (SD) sequence. In absence of ligand (orange pentagon), the aptamer part of the riboswitch (blue) is not folded. This leaves the SD sequence (green) accessible for the small ribosomal subunit (30S) and the translation occurs.
(B) Regulation of transcription termination. The aptamer domain (blue) is fused to a spacer (grey) and followed by a sequence complementary to the 3’ end of the aptamer (red). In absence of ligand, the complementary sequence binds to the aptamer and forms a terminator hairpin, which stops the RNA polymerase (RNAP). Upon ligand binding, the aptamer is formed but the terminator hairpin is not, allowing the transcription and subsequent translation of the gene. Both of these riboswitches were constructed using the synthetic theophylline aptamer. (Groher & Suess, Bioch.and Bioph. acta 2014)
“Riboswitches are also used in some higher organisms like yeast, the eukaryotic “model organism”. There are many ways to regulate gene expression in yeast with riboswitches. Some examples include control of initiating translation (the process by which proteins are created) or ribosomal shunting through allosterically controlled ribozymes, which is another mechanism for translation initiation. My (and also Adam´s) favourite method of regulation is control by splicing. To make a long story short, splicing is the step which eliminates non-coding sections of an RNA transcript (introns), of the pre-mRNA, transforming the pre-mRNA into mRNA. If the intron is not eliminated, the ribosome will not produce the right protein. This is further explained in the figure below.
“In order to regulate gene expression by splicing, some riboswitches are positioned within introns. To remove the intron, the spliceosome need to recognize specific sequences like the 5’ splice site (5’SS), the Branch point (BP) and the 3’ splice site (3’SS). If the aptamer part folds (blue in the figure below), in presence of its ligand (orange pentagon in the figure below) hiding these specific sequences, the spliceosome (U1 in the figure below) will not remove the intron and the gene will not be expressed.”
What is the future of riboswitch-based technology? Is it easy to build and use them?
Eduardo: “Based on our continuously growing knowledge on RNA biology, the development of artificial riboswitches has become a reality. However, there are several problems to take into account when doing so. The design of artificial riboswitches is based, most of the time, on its sequence similarities to known riboswitches and their predicted physicochemical characteristics. The sequence is responsible for the spatial structure the riboswitch will take, thus affecting the way it binds to its ligand, and what happens after that. That is why, apart from sequence, physicochemical properties must be taken into account when designing artificial riboswitches.
“A riboswitch structure is useless if it cannot overcome a certain energy threshold when binding its ligand (which can be any small proteins to sRNA). By overcoming this energy threshold, it is allowed to change its conformation and then regulate gene expression. There are even cases in which the objectively best – that is as modeled by chemistry data – energy values needed for the riboswitch to change its conformation may not actually be the ones that are selected. So it is important to keep that in mind, and to refine our searching and developmental methodologies.
“Based on all these facts, riboswitches have shown that they are a very important tool for synthetic biology research, and a very exciting field to work in. You will hear more about them and our work in future posts.”