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Data in support of a functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II.

Matos L, Gonçalves V, Pinto E, Laranjeira F, Prata MJ, Jordan P, Desviat LR, Pérez B, Alves S - Data Brief (2015)

Bottom Line: We have performed splicing assays for the wild-type and mutant minigenes corresponding to these substitutions.In addition, bioinformatic predictions of splicing regulatory sequence elements as well as RNA interference and overexpression experiments were conducted.The interpretation of these data and further extensive experiments into the analysis of these three mutations and also into the methodology applied to correct one of them can be found in "Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II" Matos et al. (2015) [1].

View Article: PubMed Central - PubMed

Affiliation: Research and Development Unit, Department of Human Genetics, INSA, Porto, Portugal ; Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal.

ABSTRACT
This data article contains insights into the methodology used for the analysis of three exonic mutations altering the splicing of the IDS gene: c.241C>T, c.257C>T and c.1122C>T. We have performed splicing assays for the wild-type and mutant minigenes corresponding to these substitutions. In addition, bioinformatic predictions of splicing regulatory sequence elements as well as RNA interference and overexpression experiments were conducted. The interpretation of these data and further extensive experiments into the analysis of these three mutations and also into the methodology applied to correct one of them can be found in "Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II" Matos et al. (2015) [1].

No MeSH data available.


Related in: MedlinePlus

Splicing assays for the wild-type (WT) and mutant minigenes corresponding to the IDS nucleotide changes c.257C>T and c.241C>T in exon 3, and c.1122C>T in exon 8. (A, C) Diagrams of the reporter minigenes used in the functional splicing experiments. Normal and mutated genomic IDS sequences were cloned into the pcDNA3.1-myc or pSPL3 vectors to generate the indicated minigenes. Exons are shown by boxes and introns by straight lines. For all exonic alterations, the WT and mutant regions are shown and the specific changes marked by an arrow. (B, D) Wild-type and mutant minigenes were transfected into COS-7 and Hep3B cells and the splicing pattern analyzed by RT-PCR using the indicated vector-specific primers (arrows in diagrams A and C). Minigene expression of the splicing mutations in exon 3 (c.257C>T and c.241C>T) revealed two transcripts, a predominant one with exon 2 and a mutated exon 3, and a minor transcript of smaller size in which the first 44 nucleotides of exon 3 were missing. The WT minigene produced a single transcript of normal size (B). For the synonymous c.1122C>T change in exon 8, the mutant minigene showed a single transcript lacking the last 60bp of exon 8. The WT construct produced two bands, one corresponding to the transcript with exon 8 inserted, the other to a transcript resulting from an expected splicing event between the vector splice sites (D). A diagram of the bands characterized by sequence analysis is also provided. NC – negative control; V – vector sequence.
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f0005: Splicing assays for the wild-type (WT) and mutant minigenes corresponding to the IDS nucleotide changes c.257C>T and c.241C>T in exon 3, and c.1122C>T in exon 8. (A, C) Diagrams of the reporter minigenes used in the functional splicing experiments. Normal and mutated genomic IDS sequences were cloned into the pcDNA3.1-myc or pSPL3 vectors to generate the indicated minigenes. Exons are shown by boxes and introns by straight lines. For all exonic alterations, the WT and mutant regions are shown and the specific changes marked by an arrow. (B, D) Wild-type and mutant minigenes were transfected into COS-7 and Hep3B cells and the splicing pattern analyzed by RT-PCR using the indicated vector-specific primers (arrows in diagrams A and C). Minigene expression of the splicing mutations in exon 3 (c.257C>T and c.241C>T) revealed two transcripts, a predominant one with exon 2 and a mutated exon 3, and a minor transcript of smaller size in which the first 44 nucleotides of exon 3 were missing. The WT minigene produced a single transcript of normal size (B). For the synonymous c.1122C>T change in exon 8, the mutant minigene showed a single transcript lacking the last 60bp of exon 8. The WT construct produced two bands, one corresponding to the transcript with exon 8 inserted, the other to a transcript resulting from an expected splicing event between the vector splice sites (D). A diagram of the bands characterized by sequence analysis is also provided. NC – negative control; V – vector sequence.

Mentions: Here, we performed cell-based functional splicing assays to deeper analyze the effects of two splicing mutations located in exon 3 of IDS, c.241C>T and c.257C>T and one in exon 8, c.1122C>T that were also studied in Matos et al. [1]. The pathogenic effects of these mutations are shown in Fig. 1. Also, all the data relative to oligonucleotides sequences used in the work are depicted in Table 1. Furthermore, to identify the putative SR proteins involved in the splicing regulation we have undertaken bioinformatic predictions of splicing regulatory elements (SREs) in the IDS exon 3 (where the mutations c.241C>T and c.257C>T are located) using ESEfinder 3.0 and Splicing Rainbow software (Fig. 2 and 3). Finally, we have conducted RNAi and overexpression experiments that were quantified by Real time PCR and Western blot (Table 1, Table 2 and Fig. 4).


Data in support of a functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II.

Matos L, Gonçalves V, Pinto E, Laranjeira F, Prata MJ, Jordan P, Desviat LR, Pérez B, Alves S - Data Brief (2015)

Splicing assays for the wild-type (WT) and mutant minigenes corresponding to the IDS nucleotide changes c.257C>T and c.241C>T in exon 3, and c.1122C>T in exon 8. (A, C) Diagrams of the reporter minigenes used in the functional splicing experiments. Normal and mutated genomic IDS sequences were cloned into the pcDNA3.1-myc or pSPL3 vectors to generate the indicated minigenes. Exons are shown by boxes and introns by straight lines. For all exonic alterations, the WT and mutant regions are shown and the specific changes marked by an arrow. (B, D) Wild-type and mutant minigenes were transfected into COS-7 and Hep3B cells and the splicing pattern analyzed by RT-PCR using the indicated vector-specific primers (arrows in diagrams A and C). Minigene expression of the splicing mutations in exon 3 (c.257C>T and c.241C>T) revealed two transcripts, a predominant one with exon 2 and a mutated exon 3, and a minor transcript of smaller size in which the first 44 nucleotides of exon 3 were missing. The WT minigene produced a single transcript of normal size (B). For the synonymous c.1122C>T change in exon 8, the mutant minigene showed a single transcript lacking the last 60bp of exon 8. The WT construct produced two bands, one corresponding to the transcript with exon 8 inserted, the other to a transcript resulting from an expected splicing event between the vector splice sites (D). A diagram of the bands characterized by sequence analysis is also provided. NC – negative control; V – vector sequence.
© Copyright Policy - CC BY
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4660375&req=5

f0005: Splicing assays for the wild-type (WT) and mutant minigenes corresponding to the IDS nucleotide changes c.257C>T and c.241C>T in exon 3, and c.1122C>T in exon 8. (A, C) Diagrams of the reporter minigenes used in the functional splicing experiments. Normal and mutated genomic IDS sequences were cloned into the pcDNA3.1-myc or pSPL3 vectors to generate the indicated minigenes. Exons are shown by boxes and introns by straight lines. For all exonic alterations, the WT and mutant regions are shown and the specific changes marked by an arrow. (B, D) Wild-type and mutant minigenes were transfected into COS-7 and Hep3B cells and the splicing pattern analyzed by RT-PCR using the indicated vector-specific primers (arrows in diagrams A and C). Minigene expression of the splicing mutations in exon 3 (c.257C>T and c.241C>T) revealed two transcripts, a predominant one with exon 2 and a mutated exon 3, and a minor transcript of smaller size in which the first 44 nucleotides of exon 3 were missing. The WT minigene produced a single transcript of normal size (B). For the synonymous c.1122C>T change in exon 8, the mutant minigene showed a single transcript lacking the last 60bp of exon 8. The WT construct produced two bands, one corresponding to the transcript with exon 8 inserted, the other to a transcript resulting from an expected splicing event between the vector splice sites (D). A diagram of the bands characterized by sequence analysis is also provided. NC – negative control; V – vector sequence.
Mentions: Here, we performed cell-based functional splicing assays to deeper analyze the effects of two splicing mutations located in exon 3 of IDS, c.241C>T and c.257C>T and one in exon 8, c.1122C>T that were also studied in Matos et al. [1]. The pathogenic effects of these mutations are shown in Fig. 1. Also, all the data relative to oligonucleotides sequences used in the work are depicted in Table 1. Furthermore, to identify the putative SR proteins involved in the splicing regulation we have undertaken bioinformatic predictions of splicing regulatory elements (SREs) in the IDS exon 3 (where the mutations c.241C>T and c.257C>T are located) using ESEfinder 3.0 and Splicing Rainbow software (Fig. 2 and 3). Finally, we have conducted RNAi and overexpression experiments that were quantified by Real time PCR and Western blot (Table 1, Table 2 and Fig. 4).

Bottom Line: We have performed splicing assays for the wild-type and mutant minigenes corresponding to these substitutions.In addition, bioinformatic predictions of splicing regulatory sequence elements as well as RNA interference and overexpression experiments were conducted.The interpretation of these data and further extensive experiments into the analysis of these three mutations and also into the methodology applied to correct one of them can be found in "Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II" Matos et al. (2015) [1].

View Article: PubMed Central - PubMed

Affiliation: Research and Development Unit, Department of Human Genetics, INSA, Porto, Portugal ; Department of Biology, Faculty of Sciences, University of Porto, Porto, Portugal.

ABSTRACT
This data article contains insights into the methodology used for the analysis of three exonic mutations altering the splicing of the IDS gene: c.241C>T, c.257C>T and c.1122C>T. We have performed splicing assays for the wild-type and mutant minigenes corresponding to these substitutions. In addition, bioinformatic predictions of splicing regulatory sequence elements as well as RNA interference and overexpression experiments were conducted. The interpretation of these data and further extensive experiments into the analysis of these three mutations and also into the methodology applied to correct one of them can be found in "Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II" Matos et al. (2015) [1].

No MeSH data available.


Related in: MedlinePlus