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

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].


a b s t r a c t
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 4T, c.257C 4T and c.1122C 4T.
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]. &

Data, materials and methods
The IDS gene encodes the lysosomal hydrolase iduronate-2-sulphatase, the enzyme that is deficient in the X-Linked Lysosomal Storage Disease; Mucopolysaccharidosis type II [2].
Here, we performed cell-based functional splicing assays to deeper analyze the effects of two splicing mutations located in exon 3 of IDS, c.241C4T and c.257C 4T and one in exon 8, c.1122C 4T 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.241C4T and c.257C 4T 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 (Tables 1, 2 and Fig. 4).

Oligonucleotides sequences
See Table 1.

Minigenes construction and in vitro functional splicing analysis
For the in vitro splicing analysis of the variants c.257C 4T and c.241C 4T in IDS exon 3, the respective regions of patient and healthy control genomic DNA were amplified and cloned into pcDNA3.1-myc, a modified plasmid vector (Invitrogen, Carlsbad, USA) ( Table 1 and Fig. 1A).
Also, to functionally investigate the splicing defects caused by c.1122C 4T in exon 8, wild-type (WT) and mutant minigenes were constructed in vector pSPL3 (Exon Trapping System, Life Technologies, Gibco, NY, USA) ( Table 1 and Fig. 1C). To perform the functional splicing assays, Hep3B and COS-7 cell lines (4 Â 10 5 ) were grown in 6-well plates and transfected with WT or mutant minigenes (2 μg) using 4 μl of Lipofectamine 2000 (Invitrogen, Carlsbad, USA). At 24 h post-transfection, total RNA was extracted from the cells and used as a template for cDNA synthesis. RT-PCR was then performed using vector specific primers (Table 1) and the amplified products were separated by agarose gel electrophoresis ( Fig. 1B and D).

Quantitative Real time PCR and Western blot assays to confirm overexpression and depletion of splicing factors
To confirm the predicted changes in the splicing factors SRSF2 (formerly SC35), hnRNP E1 and hnRNP E2, overexpression studies were performed using plasmids coding for them which were cotransfected in Hep3B cells with WT or mutant c.257C 4T minigenes. All transfections were performed using Lipofectamine 2000 reagent. At 48 h post-cotransfection, the cells were harvested and the transcript pattern analyzed by RT-PCR. Depletion studies were also performed to verify the predicted changes for the SRSF1 (formerly ASF/SF2) splicing factor. Hep3B cells were firstly transfected with siRNAs targeting the mRNA of SRSF1 and luciferase (control) and 24 h later transfected with the WT or mutant c.257C 4T minigenes. RT-PCR analysis was performed 48 h later. The sequences of all siRNAs are described in Table 1.  To confirm the overexpression of SRSF2, hnRNP E1 and hnRNP E2 as also the depletion of SRSF1 in whole cell lysates, quantitative Real-Time PCR (qRT-PCR) assays were performed. Relative levels of gene expression were analyzed using Taqman Universal PCR Master Mix 1x (Applied Biosystems) and the Taqman Gene Expression Kit (which includes primers and probes for each specific splicing factor gene - Table 1). The relative mRNA levels of target genes were calculated using standard curves (values ranging from 0.05 ng to 50 ng of RNA converted into cDNA). A standard curve was constructed for each target gene relating Ct values to log RNA quantities. The normalization of expression was given by the ratio between the RNA concentrations of each target gene and the endogenous gene PGK1. The relative amount of RNA was determined via the ratio of the normalized expressions of the target and control samples ( Table 2).
The depletion of the SRSF1 splicing factor was also confirmed through Western blotting analysis. The immunodetection was carried out using the primary antibodies mouse anti-SF2/ASF clone 96 from Zymed (San Francisco, CA) and anti-α-tubulin from Sigma-Aldrich (Switzerland) (Fig. 4). Table 1 Description of the different sequences used in the work: primers for PCR amplifications a ; small interfering RNA's for silencing of specific genes; probes used for real-time quantitative PCR; antisense oligonucleotide sequences.