Dissertation committee:
1. Χρήστος Κοντός, Επίκουρος Καθηγητής, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών (Επιβλέπων)
2. Ανδρέας Σκορίλας, Καθηγητής, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
3. Διαμάντης Σίδερης, Καθηγητής, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
4. Διδώ Βασιλακοπούλου, Αναπληρώτρια Καθηγήτρια, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
5. Μαργαρίτης Αυγέρης, Αναπληρωτής Καθηγητής, Ιατρική Σχολή, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
6. Αθηνά Μάρκου, Επίκουρη Καθηγήτρια, Τμήμα Χημείας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
7. Ιερώνυμος Ζωιδάκης, Επίκουρος Καθηγητής, Τμήμα Βιολογίας, Εθνικό και Καποδιστριακό Πανεπιστήμιο Αθηνών
Summary:
Non-coding RNA molecules, specifically tRNA derivatives (tDRs) and circular RNAs (circRNAs), have emerged as key players in regulating various biological processes. In the present PhD thesis, we delve into the multifaceted world of RNA regulation, focusing on 3′-tRF-CysGCA, a tRF derived from tRNACysGCA, and novel circRNAs of the BCL2L12 gene, shedding light on their roles in gene expression.
tRNA fragments (tRFs) have garnered significant attention for their potential regulatory functions. Among these, 3′-tRFs have been of particular interest due to their association with gene expression. In this study, we explored the regulatory role of 3′-tRF-CysGCA in HEK-293 cells. This tRF, deriving from the cleavage in the T-loop of tRNACysGCA, has been previously shown to interact with Argonaute proteins, implicating its role in gene expression. However, its broader impact on human cells remained unexplored. To bridge this gap, stable overexpression of 3′-tRF-CysGCA in HEK-293 cells was conducted, followed by comprehensive transcriptomics and proteomics experiments. Our findings reveal significant alterations in the cellular expression profile, while extensive bioinformatics analysis pinpointed numerous pathways affected by the deregulation of 3′-tRF-CysGCA. Additionally, reporter assays confirmed the direct interaction of this fragment with TMPO transcript variant 1 and ERGIC1, leading to changes in their expression levels. Additionally, a cell density determination assay revealed the reduced cell proliferation ratio of HEK-293 clones compared to the parental cell line. This investigation underscores the substantial regulatory role of 3′-tRF-CysGCA in shaping gene expression and its potential significance in cellular processes.
CircRNAs have recently emerged as a class of non-coding RNAs with crucial roles in gene regulation. In this study, we aimed to pioneer a high-throughput approach, employing long-read sequencing, to uncover novel circRNAs associated with the BCL2L12 gene, a member of the BCL2 family often implicated in colorectal cancer. To achieve this, we utilized multiple nested PCR assays with divergent primers targeting each BCL2L12 exon, followed by nanopore sequencing of the amplicons. Extensive data analysis, including the development of custom Perl-based algorithms, facilitated the identification of 46 novel BCL2L12 circRNAs. These circRNAs were shown to form through non-canonical back-splice sites, residing in highly similar regions of exons or introns of the primary transcripts. Furthermore, the study revealed two circRNAs containing poly(A) tracts, confirmed through Sanger sequencing and short-read sequencing. circ-BCL2L12-92, a specific circRNA identified in this study, shares miRNA-binding sites with the BCL2L12 mRNA. Silencing circ-BCL2L12-92 via siRNA transfection in HCT 116 cells resulted in the indirect downregulation of BCL2L12 mRNA, emphasizing the interconnectedness of circRNA biology with the regulation of gene expression. This observation highlights the potential role of circ-BCL2L12-92 as a modulator of BCL2L12 gene expression, uncovering a novel layer of complexity in the regulatory mechanisms governing gene expression.
In conclusion, this study offers valuable insights into the regulatory roles of 3′-tRF-CysGCA and circ-BCL2L12-92 in gene expression and unveils a plethora of novel BCL2L12 circRNAs, shedding light on the complex landscape of non-coding RNA regulation. These findings open new avenues for further research, underlining the importance of these non-coding RNA molecules in gene expression regulation.
Keywords:
non-coding RNAs, regulation of gene expression, transcriptomics, proteomics
