screening criteria
All SLC12A3 missense variants were collected from the Human Gene Mutation Database and ClinVar (October 2020). Bioinformatics software was performed to predict the effects of these variants on pre-mRNAs splicing. Firstly, analysis of BDGP (http://www.fruitfly.org) was carried out to explore the potential effects on consensus 5’ donor or 3’ acceptor sites and/or to predict the generation and/or activation of novel sites. The satisfying exons with BDGP score below 0.7 were selected to continue the analyses. Then, the Human Splicing Finder system (HSF,https://www.genomnis.com/access-hsf) was used for all missense variants in these exons to assess the impacts upon exonic splicing regulatory elements (ESEs broken and/or new ESSs creation), and variants with HSF score (ESE / ESS motifs ratio) less than -8 were selected for further minigene splicing assays. In addition, SLC12A3 variants, which located within 2 bases at the 5’ or 3’ end of the exon and significantly reduce BDGP score, were also selected as candidates for experimental analyses in this study.
3. Minigene constructions and site-directed mutagenesis
With informed consent of the subjects and the approval from the Ethics Committee of Qingdao Municipal Hospital affiliated to Qingdao University, genomic DNA was obtained from blood samples of healthy individuals using GenElute Blood Genomic DNA Extraction Kit (Sigma, NA2010). In vitro splicing assay, the fragment consisting of a target exon and flanking introns with 50-200 nucleotides is amplified by specific primers (Table S1), which contain XhoI and NheI restriction sites (XhoI : CCGC^CTCGAG; NheI : CTAG^CTAGC) and were designed by PP5 and Primer-Blast (http://www.ncbi.nlm.nih.gov/tools/primer-blast). Moreover, the truncated introns at both ends have no activation of cryptic splicing as verified by HSF. Then, PCR products purified by Gel Extraction Kit (Cwbio, China) and pSPL3 exon trapping vector were respectively digested by enzymes XhoI and NheI . This was followed by Ligation reactions with 0.2U of T4 DNA ligase (Takara, Japan) and incubation at 16℃ overnight.
Subsequently, the vectors carrying clones should be successfully transformed into DH5α competent E. coli cells (Takara, Shiga, Japan), and then proliferated in the Luria-Bertani broth. After that, an appropriate amount of the vectors was evenly spread on the IPTG/X-gal (Invitrogen, USA) coated ampicillin-Luria-Bertani agar plates at 37℃ for 16 h, and then the monoclonal colonies scattered on the plates were collected and extracted by PurePlasmid Mini Kit (Cwbio, China). The wild-type (WT) minigene was finally constructed after sequencing using forward and reverse primers and sequence analysis and alignment using Chromas 2.6.5 and Vector NTI Advance 11.
With the extension of the mutagenesis primers (Table S2) and PCR amplification, variants of interest were effectively introduced into the WT plasmids by the GeneArt™ Site-Directed Mutagenesis PLUS System (Thermo Fisher Scientific, Massachusetts, USA) according to the manufacturer’s instruction. The specific reaction system is the same as previously reported (Wang et al., 2020; Zhang et al., 2021). All constructed minigenes were further confirmed by direct sequencing.
4. Minigene splicing assay
Human epithelial kidney 293T (HEK 293T) cells and Hela cells were respectively cultured in flasks containing DMEM medium, 10% fetal bovine serum (FBS), penicillin (100 U/L) and streptomycin (100 mg/L), and incubated in a 5% CO2 incubator at 37℃. Cells need to be transferred to 12-well culture plates with an antibiotic-free medium, and after growing to about 70-80%, the transfection of each group (empty pSPL3-control (EV), pSPL3-WT and pSPL3-Mutation) was performed with OPTI-MEM® IMedium and Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) following the instruction. Forty-eight hours later, TRIzol reagent (Invitrogen, USA) was used to extract total RNA, which was produced into cDNA by RT-PCR (reverse transcription PCR) using Superscript II Reverse Transcriptase (Invitrogen Corporation, Carlsbad, CA) under the instruction booklet of manufacturer.
In order to assess the pattern of transcripts, The PCR amplification reaction was carried out with the vector-specific primers (SD6 (the forward primer: 5’-TCTGAGTCACCTGGACAACC-3’) and SA2 (the reverse primer: 5’-ATCTCAGTGGTATTTGTGAGC-3’)), as previously described (Wang et al., 2020; Zhang et al., 2021). Finally, PCR products were identified by 1.5% agarose gel electrophoresis, and each band was accurately quantified by Image J software. The percentage of exon exclusion (%) = (lower band / [lower band + upper band]) × 100. Error bars represent SEM (n=3). *P < 0.05, independent-samples T test by GraphPad Prism (Version 6.02, GraphPad Software, USA). Besides, the purification of target DNA bands was performed with a Gel Extraction Kit (CWBIO, China), followed by the sequencing and analysis of all transcripts. Only when the difference between splicing pattern and the WT minigene was observed in both cell lines was it considered significant that the variation resulted in a splicing defect.