Natural RNA Aptamers Research Articles

Variant Bacterial Riboswitches Associated with Nucleotide Hydrolase Genes Sense Nucleoside Diphosphates.

The ykkC RNA motif was a long-standing orphan riboswitch candidate that has recently been proposed to encompass at least five distinct bacterial riboswitch classes. Most ykkC RNAs belong to the subtype 1 group, which are guanidine-I riboswitches that regulate the expression of guanidine-specific carboxylase and transporter proteins. The remaining ykkC RNAs have been organized into at least four major categories called subtypes 2a-2d. Subtype 2a RNAs are riboswitches that sense the bacterial alarmone ppGpp and typically regulate amino acid biosynthesis genes. Subtype 2b riboswitches sense the purine biosynthetic intermediate PRPP and frequently partner with guanine riboswitches to regulate purine biosynthesis genes. In this study, we examined ykkC subtype 2c RNAs, which are found upstream of genes encoding hydrolase enzymes that cleave the phosphoanhydride linkages of nucleotide substrates. Subtype 2c representatives mostly recognize adenosine and cytidine 5'-diphosphate molecules in either their ribose or deoxyribose forms (ADP, dADP, CDP, and dCDP). Other nucleotide-containing compounds, especially nucleoside 5'-triphosphates, are strongly rejected by some members of this putative riboswitch class. High ligand concentrations in vivo are predicted to turn on expression of hydrolase enzymes, which presumably function to balance cellular nucleotide pools. These results further showcase the striking functional diversity derived from the structural scaffold shared among all ykkC motif RNAs, which has been adapted to sense at least five different types of natural ligands. Moreover, riboswitches for nucleoside diphosphates provide additional examples of the numerous partnerships observed between natural RNA aptamers and nucleotide-derived ligands, including metabolites, coenzymes, and signaling molecules.

Genome-wide identification of natural RNA aptamers in prokaryotes and eukaryotes

RNAs are well-suited to act as cellular sensors that detect and respond to metabolite changes in the environment, due to their ability to fold into complex structures. Here, we introduce a genome-wide strategy called PARCEL that experimentally identifies RNA aptamers in vitro, in a high-throughput manner. By applying PARCEL to a collection of prokaryotic and eukaryotic organisms, we have revealed 58 new RNA aptamers to three key metabolites, greatly expanding the list of natural RNA aptamers. The newly identified RNA aptamers exhibit significant sequence conservation, are highly structured and show an unexpected prevalence in coding regions. We identified a prokaryotic precursor tmRNA that binds vitamin B2 (FMN) to facilitate its maturation, as well as eukaryotic mRNAs that bind and respond to FMN, suggesting FMN as the second RNA-binding ligand to affect eukaryotic expression. PARCEL results show that RNA-based sensing and gene regulation is more widespread than previously appreciated in different organisms.

Structural Studies of the Purine and SAM Binding Riboswitches

Riboswitches are recently discovered genetic regulatory elements found in the 5'-untranslated regions of bacterial mRNAs that act through their ability to specifically bind small-molecule metabolites. Binding of the ligand to the aptamer domain of the riboswitch is communicated to a second domain, the expression platform, which directs transcription or translation of the mRNA. To understand this process on a molecular level, structures of three of these riboswitches bound to their cognate ligands have been solved by X-ray crystallography: the purine, thiamine pyrophosphate (TPP), and S-adenosylmethionine (SAM-I) binding aptamer domains. These studies have uncovered three common themes between the otherwise different molecules. First, the natural RNA aptamers recognize directly or indirectly almost every feature of their ligand to achieve extraordinary specificity. Second, all of these RNAs use a complex tertiary architecture to establish the binding pocket. Finally, in each case, ligand binding serves to stabilize a helix that communicates the binding event to the expression platform. Here, we discuss these properties of riboswitches in the context of the purine and SAM-I riboswitches.

An artificial riboswitch for controlling pre-mRNA splicing

Riboswitches, as previously reported, are natural RNA aptamers that regulate the expression of numerous bacterial metabolic genes in response to small molecule ligands. It has recently been shown that these RNA genetic elements are also present near the splice site junctions of plant and fungal introns, thus raising the possibility of their involvement in regulating mRNA splicing. Here it is shown for the first time that a riboswitch can be engineered to regulate pre-mRNA splicing in vitro. We show that insertion of a high-affinity theophylline binding aptamer into the 3' splice site (3' ss) region of a model pre-mRNA (AdML-Theo29AG) enables its splicing to be repressed by the addition theophylline. Our results indicate that the location of 3' ss AG within the aptamer plays a crucial role in conferring theophylline-dependent control of pre-mRNA splicing. We also show that theophylline-mediated control of pre-mRNA splicing is highly specific by first demonstrating that a small molecule ligand similar in shape and size to theophylline had no effect on the splicing of AdML-Theo29AG pre-mRNA. Second, theophylline failed to exert any influence on the splicing of a pre-mRNA that does not contain its binding site. Third, theophylline specifically blocks the step II of the splicing reaction. Finally, we provide evidence that theophylline-dependent control of pre-mRNA splicing is functionally relevant.

Riboswitches: natural SELEXion.

Advances in our knowledge of the structure and chemistry of RNA have been harnessed in the process known as SELEX to develop artificial RNA-based molecules that can act as enzymes and ligand binders performing a wide variety of functions. The discovery of riboswitches, natural RNA aptamers involved in genetic regulation, offers a basis of comparison between the artificial selection and the natural selection of structured RNAs for small-molecule recognition. The guanine riboswitch structural determination allows us to draw conclusions regarding the apparent increased complexity of the riboswitch aptamers compared to their in-vitro-selected cousins.