Julia E. Weigand

Thermodynamic characterization of an engineered tetracycline-binding riboswitch

Riboswitches reflect a novel concept in gene regulation that is particularly suited for technological adaptation. Therefore, we characterized thermodynamically the ligand binding properties of a synthetic, tetracycline (tc)-binding RNA aptamer, which regulates gene expression in a dose-dependent manner when inserted into the untranslated region of an mRNA. In vitro, one molecule of tc is bound by one molecule of partially pre-structured and conformationally homogeneous apo-RNA. The dissociation constant of 770 pM, as determined by fluorimetry, is the lowest reported so far for a small molecule-binding RNA aptamer. Additional calorimetric analysis of RNA point mutants and tc derivatives identifies functional groups crucial for the interaction and including their respective enthalpic and entropic contributions we can propose detailed structural and functional roles for certain groups. The conclusions are consistent with mutational analyses in vivo and support the hypothesis that tc-binding reinforces the structure of the RNA aptamer, preventing the scanning ribosome from melting it efficiently.

Tetracycline aptamer-controlled regulation of pre-mRNA splicing in yeast

Splicing of pre-mRNA is a critical step in mRNA maturation and disturbances cause several genetic disorders. We apply the synthetic tetracycline (tc)-binding riboswitch to establish a gene expression system for conditional tc-dependent control of pre-mRNA splicing in yeast. Efficient regulation is obtained when the aptamer is inserted close to the 5′splice site (SS) with the consensus sequence of the SS located within the aptamer stem. Structural probing indicates limited spontaneous cleavage within this stem in the absence of the ligand. Addition of tc leads to tightening of the stem and the whole aptamer structure which probably prevents recognition of the 5′SS. Combination of more then one aptamer-regulated intron increases the extent of regulation leading to highly efficient conditional gene expression systems. Our findings highlight the potential of direct RNA–ligand interaction for regulation of gene expression.

Engineered riboswitches: overview, problems and trends.

The first conditional gene expression system which employed a small molecule binding aptamer was developed several years before the discovery of natural riboswitches. With the discovery of riboswitches it became obvious that nature uses exactly the same principal of direct RNA-ligand interaction to regulate gene expression in a highly efficient, precise and fast way. In the last decade, further engineered riboswitches have been developed to control gene expression in different organisms. The successful development of new engineered riboswitches, however, is not only dependent on an innovative design but also necessitates a two step process: first, an in vitro selection which results in aptamers with high affinity binding to a desired ligand and second, a subsequent screen to identify RNAs with a desired functionality within cells. This review will give an overview of recent reports of engineered riboswitches, highlight recent developments and point out trends and problems in the field.

Highly modular structure and ligand binding by conformational capture in a minimalistic riboswitch.

Riboswitches are highly structured RNA motifs with gene regulatory activity located in the untranslated regions of mRNAs. They either modulate transcription termination or translation initiation through conformational changes triggered by direct interactions with small metabolite ligands. Many naturally occurring riboswitches are large and structurally very complex. In contrast, synthetic riboswitches—tailored gene regulatory elements for synthetic biology applications—are based on small in vitro selected RNA aptamers. Yet, despite a ligand affinity and specificity comparable to their natural counterparts only a few in vitro selected aptamers are regulatory active in vivo. Recently, Suess et al. engineered a riboswitch for the aminoglycoside antibiotic neomycin B by subjecting an in vitro SELEX-pool to an in vivo screening for gene regulatory activity in a yeastbased reporter gene assay. The resulting neomycin B and ribostamycin (Figure 1a) responsive RNA-element (N1) contains only 27 nucleotides in a bulged hairpin secondary structure (Figure 1b)—the smallest riboswitch functional in vivo identified to date. In sequence and secondary structure, N1 differs completely from an in vitro selected but regulatory inactive RNA-aptamer for the same ligand (R23). Instead it partially resembles the ribosomal A-site, the natural target for aminoglycoside antibiotics (Figure 1b). The NMR spectroscopic analysis of the N1 riboswitch complexed with ribostamycin identifies structural determinants for its regulatory activity and suggests a ligand binding mechanism based on conformational capture. Our results provide insights into the modularity of ligand binding sites in RNA and highlight structural and dynamic features N1 shares with the larger naturally occurring riboswitches as well as with other regulatory active aptamers. This knowledge may guide the future design of novel synthetic riboswitches for targeted in vivo applications. Structure of the N1–ligand complex—the “OFF”-state of the riboswitch: N1 represses gene expression upon binding to either neomycin B or the closely related but smaller ribostamycin. NMR spectra of N1 bound to either ligand (Supporting Information Figure S1) indicate that both complexes are formed with similarly high affinity and display a high degree of structural similarity suggesting that the contribution of ring IV of neomycin to the interaction is negligible. Thus, we determined the structure of the N1–ribostamycin complex, because of its superior spectral resolution for the ligand resonances, by NMR spectroscopy (see Table 1). Chemical shift assignments and coordinates have been deposited (BMRB code: 16609, pdb-code: 2kxm). The structure of ribostamycin-bound N1 consists of a continuous helical stem with canonical stacking interactions between the G5:C23 and the G9:C22 base pair despite the presence of a flexible three-nucleotide bulge (C6–U8) and a compactly folded apical hexaloop organized around a U-turn motif (U14–A16) closed by the U13:U18 base pair (Figure 1c–e). Ribostamycin rings I and II are sandwiched between the N1 major groove, in the region from G5:C23 to U13:U18 and A17 protruding from the apical loop (Figure 2). Ring III is located close to the backbone of the 3’-strand (U18 to G20). Simultaneous contacts of the ligand with the G5:C23 base pair below and G9:C22 above the bulge (Figure 2b) clamp together the lower and upper helical stem and thus enforce the uninterrupted coaxial helical stacking across the flexible C6–U8 internal bulge. The bulge itself is not interacting with the ligand. A detailed structural description of the N1– ribostamycin complex is given in the Supporting Information. A comparison of the N1–ribostamycin complex with other aminoglycoside binding RNAs reveals partial similarities to known aminoglycoside binding sub-motifs: The helical stem centered at the U10:U21 base pair is similar to the ribosomal [*] Dr. E. Duchardt-Ferner, S. R. Schmidtke, Prof. Dr. J. W hnert Institute for Molecular Biosciences, Center for Biomolecular Magnetic Resonance (BMRZ), Johann-Wolfgang-Goethe-University Frankfurt Max-von-Laue-Strasse 9, 60438 Frankfurt (Germany) Fax: (+49)69-798-29527 E-mail: woehnert@bio.uni-frankfurt.de

A fast and efficient translational control system for conditional expression of yeast genes

A new artificial regulatory system for essential genes in yeast is described. It prevents translation of target mRNAs upon tetracycline (tc) binding to aptamers introduced into their 5′UTRs. Exploiting direct RNA–ligand interaction renders auxiliary protein factors unnecessary. Therefore, our approach is strain independent and not susceptible to interferences by heterologous expressed regulatory proteins. We use a simple PCR-based strategy, which allows easy tagging of any target gene and the level of gene expression can be adjusted due to various tc aptamer-regulated promoters. As proof of concept, five differently expressed genes were targeted, two of which could not be regulated previously. In all cases, adding tc completely prevented growth and, as shown for Nop14p, rapidly abolished de novo protein synthesis providing a powerful tool for conditional regulation of yeast gene expression.

Aptamers and riboswitches: perspectives in biotechnology.

Aptamers are short, single stranded nucleic acids which bind a wide range of different ligands with extraordinary high binding affinity and specificity. The steadily increasing number of aptamers is accompanied by an expanding range of applications in biotechnology. We will describe new developments in the field including the use of aptamers for conditional gene regulation and as biosensors. In addition, we will discuss the potential of aptamers as tags to visualize RNA and protein distribution in living cells and as therapeutics. Furthermore, we will consider biotechnological applications of riboswitches for gene regulation and as drug target.

PELDOR spectroscopy reveals preorganization of the neomycin-responsive riboswitch tertiary structure.

Pulsed electron double resonance (PELDOR) spectroscopy reveals a prearranged tertiary structure of the 27 nucleotides long engineered neomycin-responsive riboswitch. Measured distances between spin labels at positions U4-U14, U4-U15, U14-U26, and U15-U26 were unchanged upon neomycin binding which implies that the global stem-loop architecture is preserved in the absence and presence of the ligand. On the basis of our results, we infer that low-temperature PELDOR data unambiguously demonstrate the existence of an enthalpically favorable set of RNA conformations ready to bind the ligand without major global rearrangement.

Hypoxia-induced alternative splicing in endothelial cells.

Background Adaptation to low oxygen by changing gene expression is vitally important for cell survival and tissue development. The sprouting of new blood vessels, initiated from endothelial cells, restores the oxygen supply of ischemic tissues. In contrast to the transcriptional response induced by hypoxia, which is mainly mediated by members of the HIF family, there are only few studies investigating alternative splicing events. Therefore, we performed an exon array for the genome-wide analysis of hypoxia-related changes of alternative splicing in endothelial cells. Methodology/Principal findings Human umbilical vein endothelial cells (HUVECs) were incubated under hypoxic conditions (1% O2) for 48 h. Genome-wide transcript and exon expression levels were assessed using the Affymetrix GeneChip Human Exon 1.0 ST Array. We found altered expression of 294 genes after hypoxia treatment. Upregulated genes are highly enriched in glucose metabolism and angiogenesis related processes, whereas downregulated genes are mainly connected to cell cycle and DNA repair. Thus, gene expression patterns recapitulate known adaptations to low oxygen supply. Alternative splicing events, until now not related to hypoxia, are shown for nine genes: six which are implicated in angiogenesis-mediated cytoskeleton remodeling (cask, itsn1, larp6, sptan1, tpm1 and robo1); one, which is involved in the synthesis of membrane-anchors (pign) and two universal regulators of gene expression (cugbp1 and max). Conclusions/Significance For the first time, this study investigates changes in splicing in the physiological response to hypoxia on a genome-wide scale. Nine alternative splicing events, until now not related to hypoxia, are reported, considerably expanding the information on splicing changes due to low oxygen supply. Therefore, this study provides further knowledge on hypoxia induced gene expression changes and presents new starting points to study the hypoxia adaptation of endothelial cells.

Mechanistic insights into an engineered riboswitch: a switching element which confers riboswitch activity

While many different RNA aptamers have been identified that bind to a plethora of small molecules only very few are capable of acting as engineered riboswitches. Even for aptamers binding the same ligand large differences in their regulatory potential were observed. We address here the molecular basis for these differences by using a set of unrelated neomycin-binding aptamers. UV melting analyses showed that regulating aptamers are thermally stabilized to a significantly higher degree upon ligand binding than inactive ones. Regulating aptamers show high ligand-binding affinity in the low nanomolar range which is necessary but not sufficient for regulation. NMR data showed that a destabilized, open ground state accompanied by extensive structural changes upon ligand binding is important for regulation. In contrast, inactive aptamers are already pre-formed in the absence of the ligand. By a combination of genetic, biochemical and structural analyses, we identified a switching element responsible for destabilizing the ligand free state without compromising the bound form. Our results explain for the first time the molecular mechanism of an engineered riboswitch.

Conformational dynamics of the tetracycline-binding aptamer

The conformational dynamics induced by ligand binding to the tetracycline-binding aptamer is monitored via stopped-flow fluorescence spectroscopy and time-correlated single photon counting experiments. The fluorescence of the ligand is sensitive to changes within the tertiary structure of the aptamer during and after the binding process. In addition to the wild-type aptamer, the mutants A9G, A13U and A50U are examined, where bases important for regulation are changed to inhibit the aptamer’s function. Our results suggest a very fast two-step-mechanism for the binding of the ligand to the aptamer that can be interpreted as a binding step followed by a reorganization of the aptamer to accommodate the ligand. Binding to the two direct contact points A13 and A50 was found to occur in the first binding step. The exchange of the structurally important base A9 for guanine induces an enormous deceleration of the overall binding process, which is mainly rooted in an enhancement of the back reaction of the first binding step by several orders of magnitude. This indicates a significant loss of tertiary structure of the aptamer in the absence of the base A9, and underlines the importance of pre-organization on the overall binding process of the tetracycline-binding aptamer.