Supervisors info:
Χρήστος Κοντός, Επίκουρος καθηγητής, Τμήμα Βιολογίας, Τομέας Βιοχημείας και Μοριακής Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Summary:
In all eukaryotic organisms, the process of splicing is carried out in the precursor translations of protein-coding genes, with the aim of generating mRNAs through the cleavage of insertions and the exon splicing. The alternative splicing phenomenon enables the generation of a variety of mRNA transcripts derived from a single gene. The complexity of the procedure justifies the strict regulation to which it is subject. However, there are several regulatory dysfunctions that may be directly related to malformations. Thus, many alternative translations produced by unexpected splice events may represent potential cancer biomarkers or therapeutic targets.
The human kallikrein gene family (KLKs) consists of 15 members and encodes the largest group of serine proteases. The KLKs are highly uniform and consist of five codons of similar size and conserved sequences. In addition, as a gene family, several alternative synergy phenomena occur, with major exons skipping, alternative 5’ and 3’ splicing sites as well as the use of alternative polyadenylation sites. Alternative translations may have abnormal expression values in various malignancies, with predictive and / or diagnostic value. It is now known that kallikrein mRNAs are targets of specific miRNAs in their 3’-UTR domains, regulating their expression.
The study of alternative stapling has been greatly expanded, notably with the use of next generation sequencing technology (NGS). In particular, the RNA sequence (RNA seq) enables the analysis of the alternative mRNA complex and the detection of new alternative transcripts, with high sensitivity and accuracy. Combined with the 3'-terminal cDNA rapid amplification technique (3’-RACE), it may offer the opportunity to study alternative polyadenylation sites and detect alternative 3-untranslated regions (3'-UTRs) in KLKs.
The aim of this thesis was to identify and identify new alternative 3'-translational regions (3'-UTRs) in the human genes of the kallikrein family, using next generation sequencing (NGS) data. To achieve this goal, bioinformatic analysis of NGS data on the selection of KLK genes and their locations, which may potentially have new alternative 3’-UTRs, was performed. Next, a nested 3’-RACE was performed using specific pairs of primers, allowing the amplification of human transcripts of KLKs in a total of 32 human cancer tissue lines. Subsequently, the identification of the new alternative 3’-UTRs discovered was carried out by Sanger sequencing.
In total, 2 new alternative 3'-UTRs were identified in translations of the KLK7 gene and one portion of a new alternative 3'-UTR in transcripts of the KLK14 gene. In addition, 7 potential
alternative 3'-UTRs, in translations of KLKs, support the results from the 3’-RACE product of electrophoresis, for which further study is needed to identify them. From the study of transcripts of the kallikrein genes, there was a large difference in the lengths and sequences of the 3’-UTRs they contain. By extension, different miRNA molecules are also observed which can potentially target alternative 3’-UTRs, as revealed by various algorithms for finding miRNA gene targeting molecules.
The new 3’-UTR variants identified are only a few of the numerous 3’-UTR alternatives that may possess the different transcripts of those genes. The detection of these regions by miRNA molecules and consequently the regulation of these genes with respect to their expression levels, especially in cases of malignancies, necessitates further study of the 3 alternative transcripts of the kallikerlin genes, as important diagnostic and / or and prognostic cancer biomarkers.
