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Absence of an intron splicing silencer in porcine Smn1 intron 7 confers immunity to the exon skipping mutation in human SMN2.

Doktor TK, Schrøder LD, Andersen HS, Brøner S, Kitewska A, Sørensen CB, Andresen BS - PLoS ONE (2014)

Bottom Line: All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon.Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice.We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.

ABSTRACT
Spinal Muscular Atrophy is caused by homozygous loss of SMN1. All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon. Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice. In this study we evaluated the potential for constructing a mini-pig animal model by introducing minimal changes in the endogenous porcine Smn1 gene to maintain the native genomic structure and regulation. We found that while a Smn2-like mutation can be introduced in the porcine Smn1 gene and can diminish the function of the ESE, it would not recapitulate the splicing pattern seen in human SMN2 due to absence of a functional ISS immediately downstream of exon 7. We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole.

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Splicing analysis of pSXN13 minigenes.A) Summary of the pSXN13 minigene and the mutations introduced in the different constructs. The hnRNP A1 binding sites within ISS-N1 have been indicated in dashed outline. Capitals indicate exonic bases. Bold italic bases in blue indicate introduced mutations. The +6C>T mutation is indicated in bold italic red. The +8G>A mutation in pigs is indicated in underlined bold. Construct numbers correspond to lane numbers in B. B) Representative RT-PCR results following transfection of Yucatan fibroblasts with minigene constructs. Inclusion expressed as a percentage is indicated in the barplot, error bars indicate standard error of mean, n = 3. Lane numbers correspond to construct numbers in A.
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pone-0098841-g002: Splicing analysis of pSXN13 minigenes.A) Summary of the pSXN13 minigene and the mutations introduced in the different constructs. The hnRNP A1 binding sites within ISS-N1 have been indicated in dashed outline. Capitals indicate exonic bases. Bold italic bases in blue indicate introduced mutations. The +6C>T mutation is indicated in bold italic red. The +8G>A mutation in pigs is indicated in underlined bold. Construct numbers correspond to lane numbers in B. B) Representative RT-PCR results following transfection of Yucatan fibroblasts with minigene constructs. Inclusion expressed as a percentage is indicated in the barplot, error bars indicate standard error of mean, n = 3. Lane numbers correspond to construct numbers in A.

Mentions: We next examined if the splicing of SMN2 exon 7 could be reestablished in a pig genomic context by introducing the exon 7+6 C>T mutation. First, we introduced the wild type pig sequence and a pig sequence with a mutation corresponding to the human +6C>T SMN2 mutation into the pSXN13 splicing reporter as previously described [19], [23], [24] and transfected Yucatan pig fibroblasts with these constructs and constructs harboring the corresponding human sequences (Fig. 2A). This showed that the pig ESE sequence has splicing enhancer function, which is slightly weaker than the corresponding human ESE sequence, and that these ESEs are functional in pig cells. We then introduced a change corresponding to the human +6C>T SMN2 mutation in the pig ESE sequence and observed that it decreased inclusion of the test exon, similar to the human SMN2 sequence (Fig. 2B). Next we disabled the previously described flanking ESS in the inserted sequences by introducing an A>C mutation [19]. These results show that the ESE in the wild type pig sequence, although functional, is slightly weaker than the ESE in the corresponding human SMN1 ESE sequence. They also show that this ESE activity in the pig sequence is decreased in the mutated pig SMN2-like sequence. Overall, the splicing patterns of pSXN13 constructs containing the pig sequences are very similar to corresponding constructs containing the human sequences (Fig. 2B), but it appears that the pig ESE is slightly weaker than the human SMN1 ESE, and that the negative effect of the +6C>T SMN2 mutation is weaker when introduced into the pig sequence.


Absence of an intron splicing silencer in porcine Smn1 intron 7 confers immunity to the exon skipping mutation in human SMN2.

Doktor TK, Schrøder LD, Andersen HS, Brøner S, Kitewska A, Sørensen CB, Andresen BS - PLoS ONE (2014)

Splicing analysis of pSXN13 minigenes.A) Summary of the pSXN13 minigene and the mutations introduced in the different constructs. The hnRNP A1 binding sites within ISS-N1 have been indicated in dashed outline. Capitals indicate exonic bases. Bold italic bases in blue indicate introduced mutations. The +6C>T mutation is indicated in bold italic red. The +8G>A mutation in pigs is indicated in underlined bold. Construct numbers correspond to lane numbers in B. B) Representative RT-PCR results following transfection of Yucatan fibroblasts with minigene constructs. Inclusion expressed as a percentage is indicated in the barplot, error bars indicate standard error of mean, n = 3. Lane numbers correspond to construct numbers in A.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4043917&req=5

pone-0098841-g002: Splicing analysis of pSXN13 minigenes.A) Summary of the pSXN13 minigene and the mutations introduced in the different constructs. The hnRNP A1 binding sites within ISS-N1 have been indicated in dashed outline. Capitals indicate exonic bases. Bold italic bases in blue indicate introduced mutations. The +6C>T mutation is indicated in bold italic red. The +8G>A mutation in pigs is indicated in underlined bold. Construct numbers correspond to lane numbers in B. B) Representative RT-PCR results following transfection of Yucatan fibroblasts with minigene constructs. Inclusion expressed as a percentage is indicated in the barplot, error bars indicate standard error of mean, n = 3. Lane numbers correspond to construct numbers in A.
Mentions: We next examined if the splicing of SMN2 exon 7 could be reestablished in a pig genomic context by introducing the exon 7+6 C>T mutation. First, we introduced the wild type pig sequence and a pig sequence with a mutation corresponding to the human +6C>T SMN2 mutation into the pSXN13 splicing reporter as previously described [19], [23], [24] and transfected Yucatan pig fibroblasts with these constructs and constructs harboring the corresponding human sequences (Fig. 2A). This showed that the pig ESE sequence has splicing enhancer function, which is slightly weaker than the corresponding human ESE sequence, and that these ESEs are functional in pig cells. We then introduced a change corresponding to the human +6C>T SMN2 mutation in the pig ESE sequence and observed that it decreased inclusion of the test exon, similar to the human SMN2 sequence (Fig. 2B). Next we disabled the previously described flanking ESS in the inserted sequences by introducing an A>C mutation [19]. These results show that the ESE in the wild type pig sequence, although functional, is slightly weaker than the ESE in the corresponding human SMN1 ESE sequence. They also show that this ESE activity in the pig sequence is decreased in the mutated pig SMN2-like sequence. Overall, the splicing patterns of pSXN13 constructs containing the pig sequences are very similar to corresponding constructs containing the human sequences (Fig. 2B), but it appears that the pig ESE is slightly weaker than the human SMN1 ESE, and that the negative effect of the +6C>T SMN2 mutation is weaker when introduced into the pig sequence.

Bottom Line: All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon.Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice.We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense M, Denmark.

ABSTRACT
Spinal Muscular Atrophy is caused by homozygous loss of SMN1. All patients retain at least one copy of SMN2 which produces an identical protein but at lower levels due to a silent mutation in exon 7 which results in predominant exclusion of the exon. Therapies targeting the splicing of SMN2 exon 7 have been in development for several years, and their efficacy has been measured using either in vitro cellular assays or in vivo small animal models such as mice. In this study we evaluated the potential for constructing a mini-pig animal model by introducing minimal changes in the endogenous porcine Smn1 gene to maintain the native genomic structure and regulation. We found that while a Smn2-like mutation can be introduced in the porcine Smn1 gene and can diminish the function of the ESE, it would not recapitulate the splicing pattern seen in human SMN2 due to absence of a functional ISS immediately downstream of exon 7. We investigated the ISS region and show here that the porcine ISS is inactive due to disruption of a proximal hnRNP A1 binding site, while a distal hnRNP A1 binding site remains functional but is unable to maintain the functionality of the ISS as a whole.

Show MeSH
Related in: MedlinePlus