Potential and Optimization of Mammalian Splice Riboswitches for the Regulation of Exon Skipping-Dependent Gene Expression and Isoform Switching within the ALOX5 Gene

Figures

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  Figure 1. Schematic illustration of controlling exon skipping at the 3′ss and 5′ss using a tetracycline riboswitch. (A) Schematic representation of the ALOX5 CDS, with the 14 exons (e1–e14) being illustrated with green boxes in relation to their respective sequence length. Intron sites are labeled with capital letters (A–M). The insertion sites for a cassette exon construct used in this study are indicated by orange (native) or blue (synthetic) arrowheads. (B) The scheme illustrates the regulation of exon skipping controlling the 3′ss using a tetracycline riboswitch. The black lines represent introns. The green boxes represent exons from the ALOX5 gene, while the red boxes illustrate synthetically introduced cassette exons containing PTCs. The riboswitch structure is colored red as it is part of the cassette exon. The inclusion or skipping of the exon during splicing is dependent on the presence of tetracycline (−/+ ligand), resulting in mRNA-containing PTCs or the correct coding sequence. (C) Scheme of the regulation of exon skipping controlling the 5′ss using a tetracycline aptamer. The fundamental concept is analogous to that employed for the regulation of the 3′ss. The riboswitch…

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  Figure 2. Synthetic ALOX5 splicing constructs show different context-dependent splicing patterns on RT-PCR and Western blot. (A) Schematic representation of the control constructs (all without riboswitch) modified to enhance exon recognition. The green boxes represent the ALOX5 CDS with the correct size given as black numbers. Nucleotide position 150 corresponds to the native EEJ of the first ALOX5 intron A. The red box represents the synthetic exon (SE) with the correct length (in nt) and origin given in the box above. The exon comprises either a repetitive 5′-CAACCAAA-3′ sequence (syn.) or the ALOX5 exon 8 sequence. Introns are illustrated by black lines and are divided into two sections according to their origin (labeled A1 and A2 for the first intron and B1 and B2 for the second intron). The intron length (in nt) and the sequence origin are given in the box above (capital letters indicate the origin of the sequence from the specific introns: HBB intron B, Dhfr introns A and C, and ALOX5 introns A and G). The complete sequences are given in Table S4. (B) The expression of the ALOX5 splicing constructs were evaluated through RT-PCR and Western blot analysis. The WT ALOX5 cDNA was used as…

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  Figure 3. Regulation of artificial ALOX5 expression using a tetracycline riboswitch to control 3′ss recognition during splicing. (A) The expression of the best regulating ALOX5 cassette-exon constructs Tc1A and Tc3A and SE8Tc1A and SE8Tc3A is shown. They were evaluated through RT-PCR and Western blot analysis. The constructs correspond to the previously tested ALOX5 control constructs (Figure 2), with the addition of a downstream integrated tetracycline riboswitch at the 3′ss of the synthetic exon (SE). Transiently transfected HEK293 cells were treated with or without 50 μM tetracycline for 24 h to test for ligand-dependent changes in ALOX5 expression. The total RNA was isolated from the cells and employed in RT-PCR using primer pairs that bind upstream and downstream of the introduced constructs. The housekeeping gene β-actin was included in the RT-PCR experiment as a loading control, with the aim of ensuring accurate quantitation of the target cDNA. The total protein was isolated from the cells and 5 μg was subjected to SDS-PAGE, followed by Western blotting and immunostaining against 5-LO (78 kDa). The protein was visualized on the SDS-PAGE gel using TCE and serve as a loading control…

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  Figure 4. Regulation of the artificial ALOX5 expression at different sites within the gene gives rise to disparate splice patterns and expression levels. (A) Schematic representation of the insertion sites of the cassette exon in the ALOX5 CDS. The distinct exons are illustrated in relation to their respective sequence lengths with green boxes. The constructs SE8TcA, SE8TcB, and SE8TcD have been inserted at native EEJs (orange arrows). The constructs 5′Tc and 3′Tc have been inserted at the synthetic EEJ with the consensus 5′-GGC/C-3′ (blue arrows). (B) Illustration of the different ALOX5 cassette-exon compositions. The green boxes represent the ALOX5 CDS, and the red box represents the synthetic exon (SE). Synthetic introns are illustrated by black lines and are divided into two sections according to their composition, labeled as A1 and A2 for intron A and as B1 and B2 for intron B. The position at which the synthetic constructs are inserted into the ALOX5 CDS is indicated by a green X and Y. The intron length (nt) and sequence origins are provided in the gray boxes. Capital letters indicate the origin of the sequence from specific introns. The length (nt) and sequence composition of the…

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  Figure 5. Regulation of artificial ALOX5 expression is enhanced by the introduction of a tetracycline-controlled cassette exon. (A) Schematic representation of the two insertion sites for double integration of cassette exons in the ALOX5 CDS. The exons are illustrated in relation to their respective sequence lengths with green boxes. The construct TcA has been inserted at the native EEJ of e1–e2 (orange arrow). The construct 3′Tc has been inserted at a synthetic EEJ with the consensus 5′-GGC/C-3′ (blue arrow). (B) Illustration of the construct with a double integrated ALOX5 cassette exon. The green boxes represent the ALOX5 CDS, and the red boxes represent synthetic exons (SE) containing PTCs. Synthetic introns are illustrated by black lines. The nt position at which the synthetic constructs are inserted into the ALOX5 CDS is indicated by black numbers. (C) The expression of all four possible combinations of the different stable ALOX5 cassette-exon constructs was evaluated through RT-PCR and Western blot analysis. Transiently transfected HEK293 cells were treated with or without 50 μM tetracycline for 24 h to test for ligand-dependent changes in the ALOX5 expression. The total RNA was…

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  Figure 6. Regulation of artificial ALOX5 expression using a tetracycline riboswitch to control 5′ss recognition during splicing and switch between 5-LO WT and isoform Δ13 expression. (A) Schematic representation of ALOX5 isoform Δ13 switch constructs. The green boxes represent the ALOX5 CDS. Synthetic introns are illustrated by black lines. The position at which the synthetic constructs are inserted into the ALOX5 CDS is indicated by black numbers and represents the e12–e13 and the e13–e14 EEJ. The intron length (nt) and sequence origins are labeled directly at the intron. HBB = human β-globin gene. (B) The expression of two ALOX5 isoform Δ13 switch constructs was evaluated through RT-PCR and Western blot analysis. The noTet construct functions as a splice control, lacking the riboswitch sequence, and exhibits the maximum attainable level of 5-LO WT expression. The ALOX5Δ13 cDNA was used as a positive control to demonstrate the maximum possible level of 5-LOΔ13 expression. Transiently transfected HEK293 cells were treated with or without 50 μM tetracycline for 24 h to test for ligand-dependent changes in the ALOX5 expression. The total RNA was isolated from the cells and employed in RT-PCR…