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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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RNA affinity pull-down experiments.A) RNA oligos spanning the ESE in SMN1 exon 7. Bases in bold indicate positions where the pig and the human sequences differ. The +6C>T mutation is indicated in bold underline. X indicates biotin. B) Western blots of protein pull-downs with oligos spanning the ESE. Two bands are seen for hnRNP A1, the upper band most likely being the alternative B splice isoform. Barplots indicate normalized band intensities for the indicated protein bands. Error bars indicate standard error of mean (n = 3). C) RNA oligos spanning the ISS in SMN1 intron 7. Bases in bold indicate positions where the pig sequence differs from the human. Mutations introduced are indicated in bold underline. X indicates biotin. The hnRNP A1 binding sites within ISS-N1 have been indicated in dashed outline. D) Western blots of protein pull-downs with oligos spanning the ISS. Barplots indicate normalized band intensities for the indicated protein bands. Error bars indicate standard error of mean (n = 3).
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pone-0098841-g004: RNA affinity pull-down experiments.A) RNA oligos spanning the ESE in SMN1 exon 7. Bases in bold indicate positions where the pig and the human sequences differ. The +6C>T mutation is indicated in bold underline. X indicates biotin. B) Western blots of protein pull-downs with oligos spanning the ESE. Two bands are seen for hnRNP A1, the upper band most likely being the alternative B splice isoform. Barplots indicate normalized band intensities for the indicated protein bands. Error bars indicate standard error of mean (n = 3). C) RNA oligos spanning the ISS in SMN1 intron 7. Bases in bold indicate positions where the pig sequence differs from the human. Mutations introduced are indicated in bold underline. X indicates biotin. The hnRNP A1 binding sites within ISS-N1 have been indicated in dashed outline. D) Western blots of protein pull-downs with oligos spanning the ISS. Barplots indicate normalized band intensities for the indicated protein bands. Error bars indicate standard error of mean (n = 3).

Mentions: To establish the interactions of the pig ESE and ISS with proteins known to bind the corresponding motifs in humans, we performed RNA-affinity purification experiments using HeLa nuclear extracts and RNA oligonucleotides with the sequences that harbor the wild type pig and human ESE and ISS motifs as well as mutated versions (Fig. 4A).


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)

RNA affinity pull-down experiments.A) RNA oligos spanning the ESE in SMN1 exon 7. Bases in bold indicate positions where the pig and the human sequences differ. The +6C>T mutation is indicated in bold underline. X indicates biotin. B) Western blots of protein pull-downs with oligos spanning the ESE. Two bands are seen for hnRNP A1, the upper band most likely being the alternative B splice isoform. Barplots indicate normalized band intensities for the indicated protein bands. Error bars indicate standard error of mean (n = 3). C) RNA oligos spanning the ISS in SMN1 intron 7. Bases in bold indicate positions where the pig sequence differs from the human. Mutations introduced are indicated in bold underline. X indicates biotin. The hnRNP A1 binding sites within ISS-N1 have been indicated in dashed outline. D) Western blots of protein pull-downs with oligos spanning the ISS. Barplots indicate normalized band intensities for the indicated protein bands. Error bars indicate standard error of mean (n = 3).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098841-g004: RNA affinity pull-down experiments.A) RNA oligos spanning the ESE in SMN1 exon 7. Bases in bold indicate positions where the pig and the human sequences differ. The +6C>T mutation is indicated in bold underline. X indicates biotin. B) Western blots of protein pull-downs with oligos spanning the ESE. Two bands are seen for hnRNP A1, the upper band most likely being the alternative B splice isoform. Barplots indicate normalized band intensities for the indicated protein bands. Error bars indicate standard error of mean (n = 3). C) RNA oligos spanning the ISS in SMN1 intron 7. Bases in bold indicate positions where the pig sequence differs from the human. Mutations introduced are indicated in bold underline. X indicates biotin. The hnRNP A1 binding sites within ISS-N1 have been indicated in dashed outline. D) Western blots of protein pull-downs with oligos spanning the ISS. Barplots indicate normalized band intensities for the indicated protein bands. Error bars indicate standard error of mean (n = 3).
Mentions: To establish the interactions of the pig ESE and ISS with proteins known to bind the corresponding motifs in humans, we performed RNA-affinity purification experiments using HeLa nuclear extracts and RNA oligonucleotides with the sequences that harbor the wild type pig and human ESE and ISS motifs as well as mutated versions (Fig. 4A).

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