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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 SMN minigenes.A) Summary of the SMN 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 underlined bases are bases that differ between humans and pigs. Bold italic bases in blue are mutations introduced. The +6C>T mutation is indicated in bold italic red. Dots within the sequence indicate a gap spanning multiple bases. Construct numbers correspond to lane numbers in B. B) 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-g003: Splicing analysis of SMN minigenes.A) Summary of the SMN 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 underlined bases are bases that differ between humans and pigs. Bold italic bases in blue are mutations introduced. The +6C>T mutation is indicated in bold italic red. Dots within the sequence indicate a gap spanning multiple bases. Construct numbers correspond to lane numbers in B. B) 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: To investigate the splicing of pig SMN exon 7, we introduced the pig ESE motif into our SMN minigene [19] and replaced 25 bp of downstream intron 7 sequence with the corresponding pig sequence (Fig. 3A). The immediate upstream intron sequence containing the poly-pyrimidine tract (PPT) and 3′splice site (3′ss) is completely identical between pig and human. When we introduced the +6C>T mutation into the pig sequence the increase in exon 7 skipping was modest (Fig. 3B) and we speculated that this could be explained by the fact, that the intron splicing silencer (ISS) sequence in pig intron 7 is different from the human ISS [25]. In humans, the ISS contains two potential hnRNP A1 binding motifs, a proximal cagcat sequence and a distal aagtga sequence, but one of these, the proximal, is abrogated in the pig (Fig. 1B, cagcat>catcat). The distal hnRNP A1 binding site seems to be strengthened in the pig sequence (aagtga>cagtga). Therefore we tested both the human ISS sequence and a mutated human ISS sequence where both potential hnRNP A1 sites are disrupted by 2A>C mutations, which have previously been shown to disrupt hnRNP A1 binding [25] (Fig 3A, constructs 3–6). Interestingly, insertion of the human ISS resulted in a modest increase in pig exon 7 exclusion (Fig. 3B), indicating that the human ISS sequence has a stronger negative effect on SMN exon 7 inclusion than the corresponding pig ISS sequence. Insertion of the the mutated human ISS resulted in a very modest decrease in pig SMN exon 7 exclusion indicating that the pig ISS has only a very modest ISS activity (Fig. 3B).


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 SMN minigenes.A) Summary of the SMN 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 underlined bases are bases that differ between humans and pigs. Bold italic bases in blue are mutations introduced. The +6C>T mutation is indicated in bold italic red. Dots within the sequence indicate a gap spanning multiple bases. Construct numbers correspond to lane numbers in B. B) 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-g003: Splicing analysis of SMN minigenes.A) Summary of the SMN 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 underlined bases are bases that differ between humans and pigs. Bold italic bases in blue are mutations introduced. The +6C>T mutation is indicated in bold italic red. Dots within the sequence indicate a gap spanning multiple bases. Construct numbers correspond to lane numbers in B. B) 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: To investigate the splicing of pig SMN exon 7, we introduced the pig ESE motif into our SMN minigene [19] and replaced 25 bp of downstream intron 7 sequence with the corresponding pig sequence (Fig. 3A). The immediate upstream intron sequence containing the poly-pyrimidine tract (PPT) and 3′splice site (3′ss) is completely identical between pig and human. When we introduced the +6C>T mutation into the pig sequence the increase in exon 7 skipping was modest (Fig. 3B) and we speculated that this could be explained by the fact, that the intron splicing silencer (ISS) sequence in pig intron 7 is different from the human ISS [25]. In humans, the ISS contains two potential hnRNP A1 binding motifs, a proximal cagcat sequence and a distal aagtga sequence, but one of these, the proximal, is abrogated in the pig (Fig. 1B, cagcat>catcat). The distal hnRNP A1 binding site seems to be strengthened in the pig sequence (aagtga>cagtga). Therefore we tested both the human ISS sequence and a mutated human ISS sequence where both potential hnRNP A1 sites are disrupted by 2A>C mutations, which have previously been shown to disrupt hnRNP A1 binding [25] (Fig 3A, constructs 3–6). Interestingly, insertion of the human ISS resulted in a modest increase in pig exon 7 exclusion (Fig. 3B), indicating that the human ISS sequence has a stronger negative effect on SMN exon 7 inclusion than the corresponding pig ISS sequence. Insertion of the the mutated human ISS resulted in a very modest decrease in pig SMN exon 7 exclusion indicating that the pig ISS has only a very modest ISS activity (Fig. 3B).

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