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.