Discovery and expression analysis of novel transcripts of the human SR-related CTD-associated factor 1 (SCAF1) gene in human cancer cells using Next-Generation Sequencing

The human SR-related CTD associated factor 1 (SCAF1) gene is a new member of the human SR (Ser/Arg-rich) superfamily of pre-mRNA splicing factors, which has been discovered and cloned by members of our lab. SCAF1 interacts with the CTD domain of the RNA polymerase II polypeptide A and is firmly involved in pre-mRNA splicing. Although it was found to be expressed widely in multiple human tissues, its mRNA levels vary a lot. The significant relation of SCAF1 with cancer has been confirmed by many studies, since SCAF1 mRNA transcript was found to be overexpressed in breast and ovarian tumors, confirming its significant prognostic value as a cancer biomarker in both these human malignancies. In this study, we describe the discovery and cloning of fifteen novel transcripts of the human SCAF1 gene (SCAF1 v.2 - v.16), using nested PCR and NGS technology. In detail, extensive bioinformatic analysis revealed that these novel SCAF1 splice variants comprise a total of nine novel alternative splicing events between the annotated exons of the gene, thus producing seven novel SCAF1 transcripts with open-reading frames, which are predicted to encode novel SCAF1 isoforms and eight novel SCAF1 transcripts with premature termination codons that are likely long non-coding RNAs. Additionally, a novel 3' UTR was discovered and cloned using nested 3' RACE and was validated with Sanger sequencing. In order to validate the NGS findings as well as to investigate the expression profile of each novel transcript, RT-PCR experiments were carried out with the use of variant-specific primers. Since SCAF1 is implicated in many human malignancies, qualifying as a potential biomarker, the quantification of the presented novel transcripts in human samples may have clinical applications in different types of cancer.