Limits...
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.

Show MeSH

Related in: MedlinePlus

RT–PCR proves influence of tc on splicing. Tc-aptamer at position a (6) and c (1), the minimer (2) and the mutant variant A9U (3) at position e grown in the absence (–) and presence (+) of tc. As controls the actin–intron without the aptamer (4) or with a mutated 5′SS (5) were analyzed. Total RNA was isolated from yeast transformed with the respective constructs, RNA of gfp and ura3 (internal control) was reverse transcribed, amplified via PCR and analyzed on a 3% agarose gel. M = DNA size marker (see Material and Methods for experimental details).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC1919484&req=5

Figure 3: RT–PCR proves influence of tc on splicing. Tc-aptamer at position a (6) and c (1), the minimer (2) and the mutant variant A9U (3) at position e grown in the absence (–) and presence (+) of tc. As controls the actin–intron without the aptamer (4) or with a mutated 5′SS (5) were analyzed. Total RNA was isolated from yeast transformed with the respective constructs, RNA of gfp and ura3 (internal control) was reverse transcribed, amplified via PCR and analyzed on a 3% agarose gel. M = DNA size marker (see Material and Methods for experimental details).

Mentions: To verify that the tc-mediated decrease in fluorescence is due to altered pre-mRNA splicing we performed RT–PCR. Figure 3 shows that the amount of spliced gfp mRNA decreases in the presence of tc [exemplarily shown for an aptamer at position a (6), position c (1) and for the minimer at position e (2)], whereas control constructs show the same ratio of spliced and unspliced mRNA in the absence and presence of tc [pWH601_A without inserted aptamer (4) or with the mutant aptamer A9U (3)]. A mutation of the consensus sequence CCAUGU leads to tc-independent inhibition of splicing. In addition, ura3 mRNA as internal control shows no influence of tc on its expression for all constructs tested (Figure 3, six lanes at the right side of both blots).Figure 3.


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

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

RT–PCR proves influence of tc on splicing. Tc-aptamer at position a (6) and c (1), the minimer (2) and the mutant variant A9U (3) at position e grown in the absence (–) and presence (+) of tc. As controls the actin–intron without the aptamer (4) or with a mutated 5′SS (5) were analyzed. Total RNA was isolated from yeast transformed with the respective constructs, RNA of gfp and ura3 (internal control) was reverse transcribed, amplified via PCR and analyzed on a 3% agarose gel. M = DNA size marker (see Material and Methods for experimental details).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: RT–PCR proves influence of tc on splicing. Tc-aptamer at position a (6) and c (1), the minimer (2) and the mutant variant A9U (3) at position e grown in the absence (–) and presence (+) of tc. As controls the actin–intron without the aptamer (4) or with a mutated 5′SS (5) were analyzed. Total RNA was isolated from yeast transformed with the respective constructs, RNA of gfp and ura3 (internal control) was reverse transcribed, amplified via PCR and analyzed on a 3% agarose gel. M = DNA size marker (see Material and Methods for experimental details).
Mentions: To verify that the tc-mediated decrease in fluorescence is due to altered pre-mRNA splicing we performed RT–PCR. Figure 3 shows that the amount of spliced gfp mRNA decreases in the presence of tc [exemplarily shown for an aptamer at position a (6), position c (1) and for the minimer at position e (2)], whereas control constructs show the same ratio of spliced and unspliced mRNA in the absence and presence of tc [pWH601_A without inserted aptamer (4) or with the mutant aptamer A9U (3)]. A mutation of the consensus sequence CCAUGU leads to tc-independent inhibition of splicing. In addition, ura3 mRNA as internal control shows no influence of tc on its expression for all constructs tested (Figure 3, six lanes at the right side of both blots).Figure 3.

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