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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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Polysome profiles of aptamer-containing mRNA indicate regulation is independent of translation initiation. (a) Polysomes were fractionated from yeast expressing both TetR and a 5-1.2-containing vYFP reporter mRNA, which were grown in the absence or presence of Dox. Polysome profiles for both the − Dox and + Dox growth conditions are shown. (b) qPCR measurements of the relative amounts of reporter mRNA within each polysome fraction, both for the − Dox and + Dox conditions. (c) qPCR measurements of relative amount of ACT1 mRNA in each polysome fraction, under − Dox and + Dox conditions. For both (b) and (c) error bars indicate the range of values for technical replicates. The data are representative of two independent, biological replicates.
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gks028-F7: Polysome profiles of aptamer-containing mRNA indicate regulation is independent of translation initiation. (a) Polysomes were fractionated from yeast expressing both TetR and a 5-1.2-containing vYFP reporter mRNA, which were grown in the absence or presence of Dox. Polysome profiles for both the − Dox and + Dox growth conditions are shown. (b) qPCR measurements of the relative amounts of reporter mRNA within each polysome fraction, both for the − Dox and + Dox conditions. (c) qPCR measurements of relative amount of ACT1 mRNA in each polysome fraction, under − Dox and + Dox conditions. For both (b) and (c) error bars indicate the range of values for technical replicates. The data are representative of two independent, biological replicates.

Mentions: Our data from cell-free translation and qPCR experiments firmly support a post-transcriptional regulatory mechanism that does not act via a decrease in mRNA levels. Therefore, using polysome analysis, we sought to define whether the aptamer–TetR interaction modulates initiation or some downstream step in the translation process. For these experiments, vYFP regulated by 5–1.2 was used as a representative target transcript. If TetR interaction with 5–1.2-vYFP mRNA predominantly inhibits translation initiation, in the absence of Dox, this should reduce 5–1.2-vYFP mRNA ribosome occupancy and lead to the transcript's accumulation in non-polysomal fractions. Conversely, disrupting the TetR–5–1.2 interaction by adding Dox would result in more efficient translation initiation and increased accumulation of 5–1.2-vYFP mRNA in polysomal fractions. However, we consistently found no significant difference in 5–1.2-vYFP mRNA ribosome occupancy between the condition where the TetR–5–1.2 interaction is intact (− Dox) or disrupted (+ Dox) (Figure 7 and Supplementary Figure S5). This suggests that either: (i) standard polysome profiling is insufficiently sensitive to detect a small but functionally important shift in ribosome occupancy that may be occurring and/or (ii) the aptamer–TetR interaction inhibits translation mainly downstream of initiation. Our polysome profiling results indicate that both the translationally repressed and actively translated 5–1.2-vYFP mRNA are similarly associated with the polysomal fractions. While polysome-associated mRNAs are generally considered to be actively translated, some of these mRNAs are known to be translationally repressed (30–34). The specific molecular details underlying repression of polysome-associated mRNA are still generally unclear, but they could involve mRNA decapping, mRNA deadenylation, altered elongation kinetics, nascent polypeptide degradation and impaired ribosome release (35). Understanding exactly how the aptamer–TetR interaction and its disruption by Dox facilitate differential partitioning of aptamer-containing target transcripts between translationally repressed and actively translated pools is an intriguing problem that will require more detailed study beyond the scope of the present work. However, such efforts could provide additional insight into regulation mechanisms downstream of translation initiation, which are increasingly being recognized to be of broad biological importance (33,35–38). Furthermore, this knowledge can further enable engineering improved versions of our presently described system for inducibly regulating protein expression.Figure 7.


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)

Polysome profiles of aptamer-containing mRNA indicate regulation is independent of translation initiation. (a) Polysomes were fractionated from yeast expressing both TetR and a 5-1.2-containing vYFP reporter mRNA, which were grown in the absence or presence of Dox. Polysome profiles for both the − Dox and + Dox growth conditions are shown. (b) qPCR measurements of the relative amounts of reporter mRNA within each polysome fraction, both for the − Dox and + Dox conditions. (c) qPCR measurements of relative amount of ACT1 mRNA in each polysome fraction, under − Dox and + Dox conditions. For both (b) and (c) error bars indicate the range of values for technical replicates. The data are representative of two independent, biological replicates.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3351163&req=5

gks028-F7: Polysome profiles of aptamer-containing mRNA indicate regulation is independent of translation initiation. (a) Polysomes were fractionated from yeast expressing both TetR and a 5-1.2-containing vYFP reporter mRNA, which were grown in the absence or presence of Dox. Polysome profiles for both the − Dox and + Dox growth conditions are shown. (b) qPCR measurements of the relative amounts of reporter mRNA within each polysome fraction, both for the − Dox and + Dox conditions. (c) qPCR measurements of relative amount of ACT1 mRNA in each polysome fraction, under − Dox and + Dox conditions. For both (b) and (c) error bars indicate the range of values for technical replicates. The data are representative of two independent, biological replicates.
Mentions: Our data from cell-free translation and qPCR experiments firmly support a post-transcriptional regulatory mechanism that does not act via a decrease in mRNA levels. Therefore, using polysome analysis, we sought to define whether the aptamer–TetR interaction modulates initiation or some downstream step in the translation process. For these experiments, vYFP regulated by 5–1.2 was used as a representative target transcript. If TetR interaction with 5–1.2-vYFP mRNA predominantly inhibits translation initiation, in the absence of Dox, this should reduce 5–1.2-vYFP mRNA ribosome occupancy and lead to the transcript's accumulation in non-polysomal fractions. Conversely, disrupting the TetR–5–1.2 interaction by adding Dox would result in more efficient translation initiation and increased accumulation of 5–1.2-vYFP mRNA in polysomal fractions. However, we consistently found no significant difference in 5–1.2-vYFP mRNA ribosome occupancy between the condition where the TetR–5–1.2 interaction is intact (− Dox) or disrupted (+ Dox) (Figure 7 and Supplementary Figure S5). This suggests that either: (i) standard polysome profiling is insufficiently sensitive to detect a small but functionally important shift in ribosome occupancy that may be occurring and/or (ii) the aptamer–TetR interaction inhibits translation mainly downstream of initiation. Our polysome profiling results indicate that both the translationally repressed and actively translated 5–1.2-vYFP mRNA are similarly associated with the polysomal fractions. While polysome-associated mRNAs are generally considered to be actively translated, some of these mRNAs are known to be translationally repressed (30–34). The specific molecular details underlying repression of polysome-associated mRNA are still generally unclear, but they could involve mRNA decapping, mRNA deadenylation, altered elongation kinetics, nascent polypeptide degradation and impaired ribosome release (35). Understanding exactly how the aptamer–TetR interaction and its disruption by Dox facilitate differential partitioning of aptamer-containing target transcripts between translationally repressed and actively translated pools is an intriguing problem that will require more detailed study beyond the scope of the present work. However, such efforts could provide additional insight into regulation mechanisms downstream of translation initiation, which are increasingly being recognized to be of broad biological importance (33,35–38). Furthermore, this knowledge can further enable engineering improved versions of our presently described system for inducibly regulating protein expression.Figure 7.

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
Related in: MedlinePlus