Inducible nuclear import by TetR aptamer-controlled 3' splice site selection.
RNA
; 27(2): 234-241, 2021 02.
Article
em En
| MEDLINE
| ID: mdl-33148600
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.
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Texto completo:
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Base de dados:
MEDLINE
Assunto principal:
Proteínas Repressoras
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Processamento Alternativo
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Sinais de Localização Nuclear
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Sítios de Splice de RNA
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Aptâmeros de Nucleotídeos
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article