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Tetracycline aptamer-controlled regulation of pre-mRNA splicing in yeast.

Weigand JE, Suess B - Nucleic Acids Res. (2007)

Bottom Line: Addition of tc leads to tightening of the stem and the whole aptamer structure which probably prevents recognition of the 5'SS.Combination of more then one aptamer-regulated intron increases the extent of regulation leading to highly efficient conditional gene expression systems.Our findings highlight the potential of direct RNA-ligand interaction for regulation of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.

ABSTRACT
Splicing of pre-mRNA is a critical step in mRNA maturation and disturbances cause several genetic disorders. We apply the synthetic tetracycline (tc)-binding riboswitch to establish a gene expression system for conditional tc-dependent control of pre-mRNA splicing in yeast. Efficient regulation is obtained when the aptamer is inserted close to the 5'splice site (SS) with the consensus sequence of the SS located within the aptamer stem. Structural probing indicates limited spontaneous cleavage within this stem in the absence of the ligand. Addition of tc leads to tightening of the stem and the whole aptamer structure which probably prevents recognition of the 5'SS. Combination of more then one aptamer-regulated intron increases the extent of regulation leading to highly efficient conditional gene expression systems. Our findings highlight the potential of direct RNA-ligand interaction for regulation of gene expression.

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Related in: MedlinePlus

Tc-aptamer regulated pre-mRNA splicing. (A) Schematic view of the vectors pWHE601_A/U. Shown is the adh promoter (PADH, black box), the gfp open reading frame with both exons E1 and E2 as gray boxes and the intron as open box. Unique restriction sites are indicated (AflII - Af, Acc65I - Ac, Bsu36I - B, ClaI - C, NheI - N, XbaI - X). The arrows indicate the transcriptional start sites with their distances to the start codon. The branch point of the intron is marked by an open triangle. (B) Model to explain tc-aptamer mediated control of splicing. The aptamer is inserted close to the 5′SS, the addition of tc facilitates the formation of a tc-aptamer complex which interferes with splicing. (C) The predicted secondary structure of the tc-aptamer is supported by structural probing (25). Important elements are indicated as stem = pedestal (P), bulge (B) and loop (L). The stem-loop 2 region of the minimer, where a GAAA tetraloop replaces nucleotides 19–36 of the aptamer, is shown top right.
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Figure 1: Tc-aptamer regulated pre-mRNA splicing. (A) Schematic view of the vectors pWHE601_A/U. Shown is the adh promoter (PADH, black box), the gfp open reading frame with both exons E1 and E2 as gray boxes and the intron as open box. Unique restriction sites are indicated (AflII - Af, Acc65I - Ac, Bsu36I - B, ClaI - C, NheI - N, XbaI - X). The arrows indicate the transcriptional start sites with their distances to the start codon. The branch point of the intron is marked by an open triangle. (B) Model to explain tc-aptamer mediated control of splicing. The aptamer is inserted close to the 5′SS, the addition of tc facilitates the formation of a tc-aptamer complex which interferes with splicing. (C) The predicted secondary structure of the tc-aptamer is supported by structural probing (25). Important elements are indicated as stem = pedestal (P), bulge (B) and loop (L). The stem-loop 2 region of the minimer, where a GAAA tetraloop replaces nucleotides 19–36 of the aptamer, is shown top right.

Mentions: We describe here a conditional gene expression system in vivo, which exploits tc-aptamer mediated inhibition of pre-mRNA splicing (Figure 1). This expands the applicability of the tc-binding aptamer to regulate gene expression. In addition, we significantly enhanced the efficiency of tc-aptamer based regulation by combining the regulatory elements that influence both translation initiation and pre-mRNA splicing.Figure 1.


Tetracycline aptamer-controlled regulation of pre-mRNA splicing in yeast.

Weigand JE, Suess B - Nucleic Acids Res. (2007)

Tc-aptamer regulated pre-mRNA splicing. (A) Schematic view of the vectors pWHE601_A/U. Shown is the adh promoter (PADH, black box), the gfp open reading frame with both exons E1 and E2 as gray boxes and the intron as open box. Unique restriction sites are indicated (AflII - Af, Acc65I - Ac, Bsu36I - B, ClaI - C, NheI - N, XbaI - X). The arrows indicate the transcriptional start sites with their distances to the start codon. The branch point of the intron is marked by an open triangle. (B) Model to explain tc-aptamer mediated control of splicing. The aptamer is inserted close to the 5′SS, the addition of tc facilitates the formation of a tc-aptamer complex which interferes with splicing. (C) The predicted secondary structure of the tc-aptamer is supported by structural probing (25). Important elements are indicated as stem = pedestal (P), bulge (B) and loop (L). The stem-loop 2 region of the minimer, where a GAAA tetraloop replaces nucleotides 19–36 of the aptamer, is shown top right.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC1919484&req=5

Figure 1: Tc-aptamer regulated pre-mRNA splicing. (A) Schematic view of the vectors pWHE601_A/U. Shown is the adh promoter (PADH, black box), the gfp open reading frame with both exons E1 and E2 as gray boxes and the intron as open box. Unique restriction sites are indicated (AflII - Af, Acc65I - Ac, Bsu36I - B, ClaI - C, NheI - N, XbaI - X). The arrows indicate the transcriptional start sites with their distances to the start codon. The branch point of the intron is marked by an open triangle. (B) Model to explain tc-aptamer mediated control of splicing. The aptamer is inserted close to the 5′SS, the addition of tc facilitates the formation of a tc-aptamer complex which interferes with splicing. (C) The predicted secondary structure of the tc-aptamer is supported by structural probing (25). Important elements are indicated as stem = pedestal (P), bulge (B) and loop (L). The stem-loop 2 region of the minimer, where a GAAA tetraloop replaces nucleotides 19–36 of the aptamer, is shown top right.
Mentions: We describe here a conditional gene expression system in vivo, which exploits tc-aptamer mediated inhibition of pre-mRNA splicing (Figure 1). This expands the applicability of the tc-binding aptamer to regulate gene expression. In addition, we significantly enhanced the efficiency of tc-aptamer based regulation by combining the regulatory elements that influence both translation initiation and pre-mRNA splicing.Figure 1.

Bottom Line: Addition of tc leads to tightening of the stem and the whole aptamer structure which probably prevents recognition of the 5'SS.Combination of more then one aptamer-regulated intron increases the extent of regulation leading to highly efficient conditional gene expression systems.Our findings highlight the potential of direct RNA-ligand interaction for regulation of gene expression.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Mikrobiologie, Friedrich-Alexander-Universität Erlangen-Nürnberg, Staudtstrasse 5, 91058 Erlangen, Germany.

ABSTRACT
Splicing of pre-mRNA is a critical step in mRNA maturation and disturbances cause several genetic disorders. We apply the synthetic tetracycline (tc)-binding riboswitch to establish a gene expression system for conditional tc-dependent control of pre-mRNA splicing in yeast. Efficient regulation is obtained when the aptamer is inserted close to the 5'splice site (SS) with the consensus sequence of the SS located within the aptamer stem. Structural probing indicates limited spontaneous cleavage within this stem in the absence of the ligand. Addition of tc leads to tightening of the stem and the whole aptamer structure which probably prevents recognition of the 5'SS. Combination of more then one aptamer-regulated intron increases the extent of regulation leading to highly efficient conditional gene expression systems. Our findings highlight the potential of direct RNA-ligand interaction for regulation of gene expression.

Show MeSH
Related in: MedlinePlus