Imaging metabolite dynamics in living cells using a Spinach-based riboswitch

Abstract

Riboswitches are natural ligand-sensing RNAs typically that are found in the 5′ UTRs of mRNA. Numerous classes of riboswitches have been discovered, enabling mRNA to be regulated by diverse and physiologically important cellular metabolites and small molecules. Here we describe Spinach riboswitches, a new class of genetically encoded metabolite sensor derived from naturally occurring riboswitches. Drawing upon the structural switching mechanism of natural riboswitches, we show that Spinach can be swapped for the expression platform of various riboswitches, allowing metabolite binding to induce Spinach fluorescence directly. In the case of the thiamine 5′-pyrophosphate (TPP) riboswitch from the Escherichia coli thiM gene encoding hydroxyethylthiazole kinase, we show that insertion of Spinach results in an RNA sensor that exhibits fluorescence upon binding TPP. This TPP Spinach riboswitch binds TPP with affinity and selectivity similar to that of the endogenous riboswitch and enables the discovery of agonists and antagonists of the TPP riboswitch using simple fluorescence readouts. Furthermore, expression of the TPP Spinach riboswitch in Escherichia coli enables live imaging of dynamic changes in intracellular TPP concentrations in individual cells. Additionally, we show that other riboswitches that use a structural mechanism similar to that of the TPP riboswitch, including the guanine and adenine riboswitches from the Bacillus subtilis xpt gene encoding xanthine phosphoribosyltransferase, and the S-adenosyl-methionine-I riboswitch from the B. subtilis yitJ gene encoding methionine synthase, can be converted into Spinach riboswitches. Thus, Spinach riboswitches constitute a novel class of RNA-based fluorescent metabolite sensors that exploit the diversity of naturally occurring ligand-binding riboswitches. 2). Binding of the target molecule induces conformational changes that reposition the fluorescent proteins. This repositioning results in changes in FRET between the fluorescent proteins, which can be imaged by microscopy in living cells (3). These sensors have provided fundamentally novel insights into the dynamic changes in calcium, cyclic nucleotides, glucose, and other molecules during cellular signaling and disease states (4).Although genetically encoded sensors are powerful tools for imaging cellular metabolites, only a small number of sensors have been created because these sensors require a protein that binds the target molecule of interest and which undergoes a targetinduced conformational change that is sufficient to induce a change in FRET. However, proteins with these properties are not readily available for most physiologically important metabolites. As a result, sensors have not been generated for many important metabolites and signaling molecules.Recently we described an alternative class of genetically encoded sensors composed of RNA (5). These sensors are based on Spinach, an "RNA mimic of green fluorescent protein" (6). Spinach is an RNA aptamer that binds fluorophores resembling the hydroxybe...

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“…These conformational switches typically activate or inhibit translation of the mRNA but can regulate other aspects of mRNA metabolism (103). Spinach riboswitches are synthetic, RNA-based, genetically encoded metabolite reporters that leverage natural riboswitch sensing mechanisms (104). Typically, when the metabolite binds a riboswitch, portions of the riboswitch are structurally remodeled, resulting in the formation of a new helix.…”

“…In a Spinach riboswitch, Spinach is inserted into the riboswitch so that the newly formed helix enables the folding of Spinach. Using this approach, naturally occurring riboswitches that bind thiamine pyrophosphate, adenine, and guanine were converted into Spinach riboswitches that fluoresce upon binding these metabolites in a concentration-dependent manner (104). …”