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Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction.

Goldfless SJ, Belmont BJ, de Paz AM, Liu JF, Niles JC - Nucleic Acids Res. (2012)

Bottom Line: Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation.By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs.In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

ABSTRACT
Sequence-specific RNA-protein interactions, though commonly used in biological systems to regulate translation, are challenging to selectively modulate. Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation. By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions. Using a reverse TetR variant, we illustrate the potential for expanding the regulatory properties of the system through protein engineering strategies.

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Systematic optimization of an aptamer variant that improves maximal expression levels and demonstration of robust regulation when an aptamer is placed in different 5′-UTR contexts. (a) Several aptamer variants generated by introducing a point mutation within Motif 1 and reducing the stem length of 5-1.2 were tested for their ability to regulate vYFP. These modifications allowed for expression level maximization while preserving regulatory range. (b) Aptamer 5-1.31 was placed within the context of several endogenous 5′-UTRs and used to control FLuc expression. In (a) and (b), numbers above the bars indicate the percent repression observed in the − Dox condition relative to the + Dox condition. In all cases, cells expressed TetR. These data show that regulation is achieved in all contexts tested.
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gks028-F6: Systematic optimization of an aptamer variant that improves maximal expression levels and demonstration of robust regulation when an aptamer is placed in different 5′-UTR contexts. (a) Several aptamer variants generated by introducing a point mutation within Motif 1 and reducing the stem length of 5-1.2 were tested for their ability to regulate vYFP. These modifications allowed for expression level maximization while preserving regulatory range. (b) Aptamer 5-1.31 was placed within the context of several endogenous 5′-UTRs and used to control FLuc expression. In (a) and (b), numbers above the bars indicate the percent repression observed in the − Dox condition relative to the + Dox condition. In all cases, cells expressed TetR. These data show that regulation is achieved in all contexts tested.

Mentions: The 5–1.2 aptamer contains an AUG start codon followed immediately by a stop codon and a second start codon that is out of frame with the first, but in frame with the downstream reporter gene. Because previous studies have demonstrated that short upstream ORFs can inhibit translation of a downstream ORF (29), we investigated whether the sequence 43AUGUGAUG50 within a predicted loop region of 5–1.2 (Figure 1a) could be primarily responsible for the translation efficiency of a downstream ORF. While keeping The rest of 5–1.2 constant, we introduced an A→G mutation at position 48 in the loop region above to generate 5–1.4d containing the sequence 43AUGUGGUG50. This change simultaneously eliminated the stop codon and the second start codon within the aptamer loop. Replacing 5–1.2 with 5–1.4d (and placing the regulated ORF in frame with the single start codon in 5–1.4d) resulted in modestly higher expression levels, but maintained TetR-dependent regulation (Figure 6a). To further increase expression of the aptamer-regulated ORF, we systematically reduced aptamer stem strength by successively eliminating base–pair interactions at the stem base while retaining sequence downstream of the initiator AUG within the aptamer (Supplementary Table S4). When we used these aptamers (5–1.30, 5–1.31, 5–1.32, 5–1.33) to control translation as described previously, we measured a large increase in maximal expression level. Furthermore, TetR-dependent regulation was preserved at the ≥80% repression level previously observed (Figure 6a). To determine the expected upper limit of expression when using 5–1.4d, we scrambled the first 25 bases of the aptamer to remove substantial predicted secondary structure (5–1.4dhalf). When used to control gene expression, this modification produced a maximal expression level comparable with that of 5–1.31, but with no TetR-dependent regulation (Figure 6a), indicating that further destabilization of the aptamer was unlikely to yield further increases in maximal expression levels. Overall, replacing 5–1.2 with 5–1.31 increases maximal expression by ∼25-fold, and with no adverse impact on the magnitude of Dox inducible, TetR-dependent regulation.Figure 6.


Direct and specific chemical control of eukaryotic translation with a synthetic RNA-protein interaction.

Goldfless SJ, Belmont BJ, de Paz AM, Liu JF, Niles JC - Nucleic Acids Res. (2012)

Systematic optimization of an aptamer variant that improves maximal expression levels and demonstration of robust regulation when an aptamer is placed in different 5′-UTR contexts. (a) Several aptamer variants generated by introducing a point mutation within Motif 1 and reducing the stem length of 5-1.2 were tested for their ability to regulate vYFP. These modifications allowed for expression level maximization while preserving regulatory range. (b) Aptamer 5-1.31 was placed within the context of several endogenous 5′-UTRs and used to control FLuc expression. In (a) and (b), numbers above the bars indicate the percent repression observed in the − Dox condition relative to the + Dox condition. In all cases, cells expressed TetR. These data show that regulation is achieved in all contexts tested.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351163&req=5

gks028-F6: Systematic optimization of an aptamer variant that improves maximal expression levels and demonstration of robust regulation when an aptamer is placed in different 5′-UTR contexts. (a) Several aptamer variants generated by introducing a point mutation within Motif 1 and reducing the stem length of 5-1.2 were tested for their ability to regulate vYFP. These modifications allowed for expression level maximization while preserving regulatory range. (b) Aptamer 5-1.31 was placed within the context of several endogenous 5′-UTRs and used to control FLuc expression. In (a) and (b), numbers above the bars indicate the percent repression observed in the − Dox condition relative to the + Dox condition. In all cases, cells expressed TetR. These data show that regulation is achieved in all contexts tested.
Mentions: The 5–1.2 aptamer contains an AUG start codon followed immediately by a stop codon and a second start codon that is out of frame with the first, but in frame with the downstream reporter gene. Because previous studies have demonstrated that short upstream ORFs can inhibit translation of a downstream ORF (29), we investigated whether the sequence 43AUGUGAUG50 within a predicted loop region of 5–1.2 (Figure 1a) could be primarily responsible for the translation efficiency of a downstream ORF. While keeping The rest of 5–1.2 constant, we introduced an A→G mutation at position 48 in the loop region above to generate 5–1.4d containing the sequence 43AUGUGGUG50. This change simultaneously eliminated the stop codon and the second start codon within the aptamer loop. Replacing 5–1.2 with 5–1.4d (and placing the regulated ORF in frame with the single start codon in 5–1.4d) resulted in modestly higher expression levels, but maintained TetR-dependent regulation (Figure 6a). To further increase expression of the aptamer-regulated ORF, we systematically reduced aptamer stem strength by successively eliminating base–pair interactions at the stem base while retaining sequence downstream of the initiator AUG within the aptamer (Supplementary Table S4). When we used these aptamers (5–1.30, 5–1.31, 5–1.32, 5–1.33) to control translation as described previously, we measured a large increase in maximal expression level. Furthermore, TetR-dependent regulation was preserved at the ≥80% repression level previously observed (Figure 6a). To determine the expected upper limit of expression when using 5–1.4d, we scrambled the first 25 bases of the aptamer to remove substantial predicted secondary structure (5–1.4dhalf). When used to control gene expression, this modification produced a maximal expression level comparable with that of 5–1.31, but with no TetR-dependent regulation (Figure 6a), indicating that further destabilization of the aptamer was unlikely to yield further increases in maximal expression levels. Overall, replacing 5–1.2 with 5–1.31 increases maximal expression by ∼25-fold, and with no adverse impact on the magnitude of Dox inducible, TetR-dependent regulation.Figure 6.

Bottom Line: Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation.By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs.In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Engineering, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.

ABSTRACT
Sequence-specific RNA-protein interactions, though commonly used in biological systems to regulate translation, are challenging to selectively modulate. Here, we demonstrate the use of a chemically-inducible RNA-protein interaction to regulate eukaryotic translation. By genetically encoding Tet Repressor protein (TetR)-binding RNA elements into the 5'-untranslated region (5'-UTR) of an mRNA, translation of a downstream coding sequence is directly controlled by TetR and tetracycline analogs. In endogenous and synthetic 5'-UTR contexts, this system efficiently regulates the expression of multiple target genes, and is sufficiently stringent to distinguish functional from non-functional RNA-TetR interactions. Using a reverse TetR variant, we illustrate the potential for expanding the regulatory properties of the system through protein engineering strategies.

Show MeSH