An artificial riboswitch for controlling pre-mRNA splicing

Abstract

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 0 splice site (3 0 ss) region of a model pre-mRNA (AdMLTheo29AG) enables its splicing to be repressed by the addition theophylline. Our results indicate that the location of 3 0 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.

Mechanistic Control of Carcinoembryonic Antigen-related Cell Adhesion Molecule-1 (CEACAM1) Splice Isoforms by the Heterogeneous Nuclear Ribonuclear Proteins hnRNP L, hnRNP A1, and hnRNP M

“…Separation of splicing-related complexes were assembled in HeLa nuclear extracts, 12.5-l total volume reactions, containing exon 7 RNA (10,000 cpm/l) incubated in the absence or presence of unlabeled NS competitor RNA or unlabeled exon 7 RNA under conditions that support in vitro splicing as described previously (37). Reactions were incubated at 30°C for 15 min.…”

Reengineering orthogonally selective riboswitches

“…Notably, insertion of aptamers into the 5′-UTRs of bacterial or eukaryotic mRNAs (9)(10)(11)(12), splice sites within introns of eukaryotic pre-mRNA (13,14), or coupling of aptamers to ribozymes (15,16) have shown significant potential for small-molecule modulation of gene expression. However, there are currently very few aptamers, generated by in vitro selection, that are selective for ligands with desirable physicochemical and pharmacokinetic properties (8).…”

Rational design of artificial riboswitches based on ligand-dependent modulation of internal ribosome entry in wheat germ extract and their applications as label-free biosensors

“…A first design of the ligand-inducible IRES-mediated translation system was achieved by using a theophylline aptamer, which was obtained by an in vitro selection method (Jenison et al 1994) and has been used for constructing many artificial riboswitches (Harvey et al 2002;Suess et al 2003;Desai and Gallivan 2004;Kim et al 2005;Ogawa andMaeda 2007, 2008a,b;Win and Smolke 2007;Ogawa 2009) because of its high binding and discriminating ability for its ligand, theophylline. The length of a 39 part of MS hybridizing to the aaIRES was fixed to 4-mer (59-AGAC-39).…”

“…However, whereas many in vivo applications of artificial riboswitches in both prokaryotes and eukaryotes have been reported thus far (Werstuck and Green 1998;Grate and Wilson 2001;Harvey et al 2002;Suess et al 2003Suess et al , 2004Desai and Gallivan 2004;Kim et al 2005;Nomura and Yokobayashi 2007;Weigand and Suess 2007;Win and Smolke 2007;Ogawa and Maeda 2008a;Wieland and Hartig 2008;Weigand et al 2008), there have been few examples of their applications as in vitro biosensors. I and Maeda have previously reported two types of prokaryotic riboswitch-based in vitro biosensors that were rationally designed using aptazymes, which are functional RNA and cleave themselves in response to their specific ligand molecules (Ogawa andMaeda 2007, 2008b).…”

“…I therefore have decided to establish a method for rationally constructing pt-ON-riboswitches in vitro by the use of aptamers without ribozymes. Moreover, I chose to design eukaryotic ribozymefree aptamer-based pt-ON-riboswitches because there has been no example of those obtained, even via selection methods, in contrast to prokaryotic ribozyme-free aptamer-based pt-ON-riboswitches selected in vivo (Desai and Gallivan 2004;Nomura and Yokobayashi 2007;Muranaka et al 2009) or eukaryotic ribozyme-free aptamer-based pt-OFF-riboswitches (down-regulation-type) (Werstuck and Green 1998;Grate and Wilson 2001;Harvey et al 2002;Suess et al 2003;Kim et al 2005;Weigand and Suess 2007;Weigand et al 2008).…”