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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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Double-intron constructs enhance regulatory efficiency. (A) Relative GFP activity measured in the absence (closed) and presence (open bars) of 250 µM tc. Regulation efficiency determined as the ratio of fluorescence without and with tc is given by the numbers above the bars within the blot. (B) Schematic view of the various constructs. The mRNA is shown as black line, the cap structure as black dot and the exons of the gfp open reading frame as gray boxes. The distance between two aptamers is given in nt.
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Figure 4: Double-intron constructs enhance regulatory efficiency. (A) Relative GFP activity measured in the absence (closed) and presence (open bars) of 250 µM tc. Regulation efficiency determined as the ratio of fluorescence without and with tc is given by the numbers above the bars within the blot. (B) Schematic view of the various constructs. The mRNA is shown as black line, the cap structure as black dot and the exons of the gfp open reading frame as gray boxes. The distance between two aptamers is given in nt.

Mentions: Previous studies showed that riboswitch activity of the tc-aptamer can inhibit different steps of translation initiation. It interferes with initial binding of the 43S subunit when located close to the cap structure but also inhibits 80S formation when inserted at a more downstream position, probably by impeding the scanning ribosome (15). Thereby, a 6-fold regulation was obtained when inserting the aptamer directly adjacent to the start codon. We speculated that combining translation with splicing control might further increase regulation. We used the intron construct with the highest regulation factor (minimer at position e with the 9 nt long stem) and combined it with an aptamer inserted directly in front of the start codon resulting in D1 (Figure 4). The construct shows a slightly reduced regulation compared to the single intron construct, however the two aptamers are separated by only 12 nt so that an interaction cannot be excluded. Therefore, we constructed a gfp gene with an intron at a position further downstream (at amino acid 47) resulting in D2. Both double constructs result in a remarkable increase in regulation (11- and 29-fold, respectively), however, regulation is accompanied by a more pronounced drop in basal activity.Figure 4.


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

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

Double-intron constructs enhance regulatory efficiency. (A) Relative GFP activity measured in the absence (closed) and presence (open bars) of 250 µM tc. Regulation efficiency determined as the ratio of fluorescence without and with tc is given by the numbers above the bars within the blot. (B) Schematic view of the various constructs. The mRNA is shown as black line, the cap structure as black dot and the exons of the gfp open reading frame as gray boxes. The distance between two aptamers is given in nt.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Double-intron constructs enhance regulatory efficiency. (A) Relative GFP activity measured in the absence (closed) and presence (open bars) of 250 µM tc. Regulation efficiency determined as the ratio of fluorescence without and with tc is given by the numbers above the bars within the blot. (B) Schematic view of the various constructs. The mRNA is shown as black line, the cap structure as black dot and the exons of the gfp open reading frame as gray boxes. The distance between two aptamers is given in nt.
Mentions: Previous studies showed that riboswitch activity of the tc-aptamer can inhibit different steps of translation initiation. It interferes with initial binding of the 43S subunit when located close to the cap structure but also inhibits 80S formation when inserted at a more downstream position, probably by impeding the scanning ribosome (15). Thereby, a 6-fold regulation was obtained when inserting the aptamer directly adjacent to the start codon. We speculated that combining translation with splicing control might further increase regulation. We used the intron construct with the highest regulation factor (minimer at position e with the 9 nt long stem) and combined it with an aptamer inserted directly in front of the start codon resulting in D1 (Figure 4). The construct shows a slightly reduced regulation compared to the single intron construct, however the two aptamers are separated by only 12 nt so that an interaction cannot be excluded. Therefore, we constructed a gfp gene with an intron at a position further downstream (at amino acid 47) resulting in D2. Both double constructs result in a remarkable increase in regulation (11- and 29-fold, respectively), however, regulation is accompanied by a more pronounced drop in basal activity.Figure 4.

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