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Trans-splicing improvement by the combined application of antisense strategies.

Koller U, Hainzl S, Kocher T, Hüttner C, Klausegger A, Gruber C, Mayr E, Wally V, Bauer JW, Murauer EM - Int J Mol Sci (2015)

Bottom Line: We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency.Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level.This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. u.koller@salk.at.

ABSTRACT
Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

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Related in: MedlinePlus

Detection of full-length GFP restoration by western blot analysis. The corresponding band for GFP is visible at a size of ~27 kDa on the nitrocellulose membrane after immunostaining. The amount of detected GFP increases with the amount of added AS RNA-13 (lanes 1–6: 0, 2–6 µg AS RNA-13). Analysis of the intensity of GFP signal revealed an up to eight-fold (relative quantification of GFP expression was measured using the Image Lab 3.0.1 software (Bio-Rad, Hercules, CA, USA) up-regulation after the addition of 6 µg AS RNA-13 expressing plasmids. Annexin I was included as loading control in the experiment visible at a size of 37 kDa on the nitrocellulose membrane.
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ijms-16-01179-f004: Detection of full-length GFP restoration by western blot analysis. The corresponding band for GFP is visible at a size of ~27 kDa on the nitrocellulose membrane after immunostaining. The amount of detected GFP increases with the amount of added AS RNA-13 (lanes 1–6: 0, 2–6 µg AS RNA-13). Analysis of the intensity of GFP signal revealed an up to eight-fold (relative quantification of GFP expression was measured using the Image Lab 3.0.1 software (Bio-Rad, Hercules, CA, USA) up-regulation after the addition of 6 µg AS RNA-13 expressing plasmids. Annexin I was included as loading control in the experiment visible at a size of 37 kDa on the nitrocellulose membrane.

Mentions: To confirm the accurate fusion of the two GFP split parts by RNA trans-splicing on protein level, we performed western blot analysis 48 h post-transfection. We detected full-length GFP (26.9 kDa), resulting from successful trans-splicing. As shown in Figure 4, the intensity of the full-length GFP band was much higher after AS RNA-13 administration. This experiment stressed the accurate fusion of both GFP split parts by RNA trans-splicing and underlined the possibility to enhance the trans-splicing efficiency by the addition of AS RNA-13, which probably interferes with cis-splicing elements on the COL7A1-MG.


Trans-splicing improvement by the combined application of antisense strategies.

Koller U, Hainzl S, Kocher T, Hüttner C, Klausegger A, Gruber C, Mayr E, Wally V, Bauer JW, Murauer EM - Int J Mol Sci (2015)

Detection of full-length GFP restoration by western blot analysis. The corresponding band for GFP is visible at a size of ~27 kDa on the nitrocellulose membrane after immunostaining. The amount of detected GFP increases with the amount of added AS RNA-13 (lanes 1–6: 0, 2–6 µg AS RNA-13). Analysis of the intensity of GFP signal revealed an up to eight-fold (relative quantification of GFP expression was measured using the Image Lab 3.0.1 software (Bio-Rad, Hercules, CA, USA) up-regulation after the addition of 6 µg AS RNA-13 expressing plasmids. Annexin I was included as loading control in the experiment visible at a size of 37 kDa on the nitrocellulose membrane.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307297&req=5

ijms-16-01179-f004: Detection of full-length GFP restoration by western blot analysis. The corresponding band for GFP is visible at a size of ~27 kDa on the nitrocellulose membrane after immunostaining. The amount of detected GFP increases with the amount of added AS RNA-13 (lanes 1–6: 0, 2–6 µg AS RNA-13). Analysis of the intensity of GFP signal revealed an up to eight-fold (relative quantification of GFP expression was measured using the Image Lab 3.0.1 software (Bio-Rad, Hercules, CA, USA) up-regulation after the addition of 6 µg AS RNA-13 expressing plasmids. Annexin I was included as loading control in the experiment visible at a size of 37 kDa on the nitrocellulose membrane.
Mentions: To confirm the accurate fusion of the two GFP split parts by RNA trans-splicing on protein level, we performed western blot analysis 48 h post-transfection. We detected full-length GFP (26.9 kDa), resulting from successful trans-splicing. As shown in Figure 4, the intensity of the full-length GFP band was much higher after AS RNA-13 administration. This experiment stressed the accurate fusion of both GFP split parts by RNA trans-splicing and underlined the possibility to enhance the trans-splicing efficiency by the addition of AS RNA-13, which probably interferes with cis-splicing elements on the COL7A1-MG.

Bottom Line: We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency.Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level.This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

View Article: PubMed Central - PubMed

Affiliation: Department of Dermatology and EB House Austria, Paracelsus Medical University, Salzburg 5020, Austria. u.koller@salk.at.

ABSTRACT
Spliceosome-mediated RNA trans-splicing has become an emergent tool for the repair of mutated pre-mRNAs in the treatment of genetic diseases. RNA trans-splicing molecules (RTMs) are designed to induce a specific trans-splicing reaction via a binding domain for a respective target pre-mRNA region. A previously established reporter-based screening system allows us to analyze the impact of various factors on the RTM trans-splicing efficiency in vitro. Using this system, we are further able to investigate the potential of antisense RNAs (AS RNAs), presuming to improve the trans-splicing efficiency of a selected RTM, specific for intron 102 of COL7A1. Mutations in the COL7A1 gene underlie the dystrophic subtype of the skin blistering disease epidermolysis bullosa (DEB). We have shown that co-transfections of the RTM and a selected AS RNA, interfering with competitive splicing elements on a COL7A1-minigene (COL7A1-MG), lead to a significant increase of the RNA trans-splicing efficiency. Thereby, accurate trans-splicing between the RTM and the COL7A1-MG is represented by the restoration of full-length green fluorescent protein GFP on mRNA and protein level. This mechanism can be crucial for the improvement of an RTM-mediated correction, especially in cases where a high trans-splicing efficiency is required.

Show MeSH
Related in: MedlinePlus