Molecular Sciences III.
Gabriella Orosz1, Luca Szabó1, Szanna Bereti1, Veronika Zámbó1, Miklós Csala1, Éva Kereszturi1
1 Department of Molecular Biology, Semmelweis University, H-1085 Budapest, Hungary
Alternative splicing (AS) is crucial for human gene expression regulation, which plays a decisive role in expanding the diversity of functional proteins. Cancer cells have general alterations in the splicing process that may have prognostic, predictive and therapeutic values. The expression and AS of stearoyl-CoA desaturase-5 (SCD5) is considered as a promising tumor type-specific marker.
Our work was aimed at finding out whether the two transcript variants (TVs) of SCD5s (A and B) are indeed transcribed into mRNA and at what rate they are expressed in different human tissues. A further aim was to investigate the mechanisms of SCD5 splicing and the impact of single nucleotide variants (SNVs) affecting the sequences of donor and acceptor sites relevant for AS.
The expression of SCD5 TVs was detected by RT-PCR and qPCR. The NetGene prediction program was used to investigate in silico the intron editing differences between the A and B forms of SCD5. For in vitro analysis, a specific SCD5 minigene construct was generated and assayed by transient transfection in HEK293T cells. The splice site SNVs were generated by site-directed mutagenesis and their effects were monitored both at the mRNA and protein levels by qPCR and immunoblot, respectively.
Both TVs of SCD5 are equally expressed in the human tissues studied, with the highest expression in the brain, pancreas and gonads. We found that the SCD5A is present in all tissues at least one order of magnitude higher than the B form. The NetGene program predicted a significantly lower probability of a splice site at donor site B, compared to donor site A of SCD5A. This was experimentally verified at both mRNA and protein level using the self-designed SCD5 minigene construct. The reason for this unequal distribution was identified in the poorer recognition of the SCD5B splicing acceptor sequence. The SCD5 minigene construct was also used to investigate the effect of SNVs affecting the donor and acceptor recognition site sequences on SCD5 distribution. Nucleotide variations affecting TV-specific acceptor sites strongly modified or even reversed the relative expression levels of SCD5A and SCD5B.
The strength of crucial splice sites and the impact of their SNVs on AS can greatly influence prognosis and offer the promise of novel and personalized approaches to cancer treatment.
This work was supported by NKFIH grant (FK138115).