Inducible nuclear import by TetR aptamer-controlled 3′ splice site selection

Abstract

Correct cellular localization is essential for the function of many eukaryotic proteins and hence cell physiology. Here, we present a synthetic genetic device that allows the control of nuclear and cytosolic localization based on controlled alternative splicing in human cells. The device is based on the fact that an alternative 3 ′ ′ ′ ′ ′ splice site is located within a TetR aptamer that in turn is positioned between the branch point and the canonical splice site. The novel splice site is only recognized when the TetR repressor is bound. Addition of doxycycline prevents TetR aptamer binding and leads to recognition of the canonical 3 ′ ′ ′ ′ ′ splice site. It is thus possible to produce two independent splice isoforms. Since the terminal loop of the aptamer may be replaced with any sequence of choice, one of the two isoforms may be extended by the respective sequence of choice depending on the presence of doxycycline. In a proof-of-concept study, we fused a nuclear localization sequence to a cytosolic target protein, thus directing the protein into the nucleus. However, the system is not limited to the control of nuclear localization. In principle, any target sequence can be integrated into the aptamer, allowing not only the production of a variety of different isoforms on demand, but also to study the function of mislocalized proteins. Moreover, it also provides a valuable tool for investigating the mechanism of alternative splicing in human cells.

Riboswitches for Controlled Expression of Therapeutic Transgenes Delivered by Adeno-Associated Viral Vectors

“…As with other non-self protein-mediated expression control systems however, TetR immunogenicity and the size of its expression cassette may limit use in AAV. Even so, the recent use of this system by Mol et al to control inclusion of an alternative exon with a nuclear localization sequence instead of a premature stop codon points toward a wider array of applications for riboswitches which mediate splicing [74]; combining riboswitches with orthogonal ligands could be used to control both expression and function of transgene products.…”

Sensing intracellular signatures with synthetic mRNAs

“…In TSD, the TetR aptamer was inserted into an intron close to a 5’ splice site (5’SS) such that TetR-aptamer binding inhibits spliceosome assembly, consequently leading to intron retention. A more recent study employed the TetR aptamer to control switching between two alternative splice isoforms depending on the binding of TetR to the aptamer [ 57 ]. In this system, an intron harbours two 3’ splices sites (3’SSs), and TetR binding to the aptamer in the intron alters 3’SSs selection.…”

A Novel RNA Aptamer as Synthetic Inducer of DasR Controlled Transcription

“…25,26 This allowed an alternative activator of TetR-controlled transcription but also for the control of mRNA splicing and translation, by inserting the aptamer either close to splice sites or within the 5′ UTR. 27,28 Hence, the TetR-binding aptamer allowed for significant extension of the already important tet on/off system. As this system works so impressively well, we aim to enlarge the toolbox of synthetic biology with further aptamer/repressor pairs to enable more sophisticated applications.…”

“…To add a further aspect to this system, we have selected a TetR binding RNA aptamer by an in vitro process called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). , RNA aptamers are single-stranded nucleic acids that can fold into a complex three-dimensional structure that enables them to bind their target molecule with high affinity and specificity. The TetR-binding RNA aptamer is capable of recognizing TetR thereby blocking its DNA-binding ability in vitro as well as in vivo . , This allowed an alternative activator of TetR-controlled transcription but also for the control of mRNA splicing and translation, by inserting the aptamer either close to splice sites or within the 5′ UTR. , Hence, the TetR-binding aptamer allowed for significant extension of the already important tet on/off system. As this system works so impressively well, we aim to enlarge the toolbox of synthetic biology with further aptamer/repressor pairs to enable more sophisticated applications.…”