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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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Related in: MedlinePlus

Genomic structure of SMN1 genes in humans, pigs and mice.A) SMN1 pre-mRNA transcripts expressed in humans, pigs and mice. Exons included in transcripts are numbered according to historical nomenclature. Presence of pseudoexons in processed introns are indicated in dashed outline and coloured according to the species expressing transcripts including these exons. Introns are drawn to scale and indicated as lines, exons are not drawn to scale and are indicated as boxes. Start-codon is indicated by ATG and stop-codon by TAA. B) The start-sequence of intron 7 in humans, pigs and mice. Bases that differ from the human sequence are indicated in bold underline. The location of the human ISS is indicated in shade.
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pone-0098841-g001: Genomic structure of SMN1 genes in humans, pigs and mice.A) SMN1 pre-mRNA transcripts expressed in humans, pigs and mice. Exons included in transcripts are numbered according to historical nomenclature. Presence of pseudoexons in processed introns are indicated in dashed outline and coloured according to the species expressing transcripts including these exons. Introns are drawn to scale and indicated as lines, exons are not drawn to scale and are indicated as boxes. Start-codon is indicated by ATG and stop-codon by TAA. B) The start-sequence of intron 7 in humans, pigs and mice. Bases that differ from the human sequence are indicated in bold underline. The location of the human ISS is indicated in shade.

Mentions: In humans, exon 7 is followed by a 444 bp long intron but in other animals, such as the mouse, intron 7 is much longer and the final exon, exon 8, is completely different from the human sequence. In pigs, however, exon 8 is a small 26 bp long exon located 8.9 kb downstream from exon 7 and is then followed by a 3 kb long intron 8 after which the porcine Smn1 exon 9 begins (Fig. 1A). Despite the significant difference in intron lengths, porcine exon 9 and murine exon 8 seem to be homologous. One striking observation though, is the overall similarity between human SMN1/SMN2 and porcine Smn1 in terms of exon-intron structure, with the exception of the 3′ end of the transcripts, while the intron lengths are very different in murine Smn1.


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)

Genomic structure of SMN1 genes in humans, pigs and mice.A) SMN1 pre-mRNA transcripts expressed in humans, pigs and mice. Exons included in transcripts are numbered according to historical nomenclature. Presence of pseudoexons in processed introns are indicated in dashed outline and coloured according to the species expressing transcripts including these exons. Introns are drawn to scale and indicated as lines, exons are not drawn to scale and are indicated as boxes. Start-codon is indicated by ATG and stop-codon by TAA. B) The start-sequence of intron 7 in humans, pigs and mice. Bases that differ from the human sequence are indicated in bold underline. The location of the human ISS is indicated in shade.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098841-g001: Genomic structure of SMN1 genes in humans, pigs and mice.A) SMN1 pre-mRNA transcripts expressed in humans, pigs and mice. Exons included in transcripts are numbered according to historical nomenclature. Presence of pseudoexons in processed introns are indicated in dashed outline and coloured according to the species expressing transcripts including these exons. Introns are drawn to scale and indicated as lines, exons are not drawn to scale and are indicated as boxes. Start-codon is indicated by ATG and stop-codon by TAA. B) The start-sequence of intron 7 in humans, pigs and mice. Bases that differ from the human sequence are indicated in bold underline. The location of the human ISS is indicated in shade.
Mentions: In humans, exon 7 is followed by a 444 bp long intron but in other animals, such as the mouse, intron 7 is much longer and the final exon, exon 8, is completely different from the human sequence. In pigs, however, exon 8 is a small 26 bp long exon located 8.9 kb downstream from exon 7 and is then followed by a 3 kb long intron 8 after which the porcine Smn1 exon 9 begins (Fig. 1A). Despite the significant difference in intron lengths, porcine exon 9 and murine exon 8 seem to be homologous. One striking observation though, is the overall similarity between human SMN1/SMN2 and porcine Smn1 in terms of exon-intron structure, with the exception of the 3′ end of the transcripts, while the intron lengths are very different in murine Smn1.

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