| Summary: | In less than a decade since the sequencing of the human genome, it has become clear that over 85% of the genome encodes RNA transcripts that do not translate into proteins. These transcripts are also known as non-coding RNAs (ncRNAs) and were previously divided into housekeeping and regulatory ncRNAs. Among the most prominent housekeeping examples of ncRNAs that we now known that exhibit essential regulatory roles are the transfer RNA (tRNA), ribosomal RNA (rRNA), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), RNase P RNA, and telomerase RNA (Zhang et al., 2019). In addition, the ncRNA transcriptome has been recognized as a major regulome that includes microRNAs (miRNAs), long non-coding RNAs (lncRNAs), PiWi RNAs (piRNAs), tRNA-derived fragments (tRFs), vault RNAs etc., and controls crucial metabolic processes in the cell (Morris and Mattick, 2014; Schimmel, 2018; Lorenzi et al., 2021). Moreover, they have emerged as important biomarkers of homeostasis at the epigenetic, transcriptional, post-transcriptional and translational level (Schimmel, 2018; Slack and Chinnaiyan, 2019). Upon transcription, all ncRNAs undergo numerous maturation steps which are further mediated by important enzymes, ribonucleoprotein complexes or scaffold proteins of known or putative function, generally known as RNA binding proteins (RBPs). In many cases, the interactions between protein factors and ncRNAs can determine not only the function and the fate of an RNA molecule, but also can affect transcription and translation rates, overall. Elucidation of the role of ncRNAs-interacting proteins is critical to understand the hardcore biology and molecular functions of ncRNAs, to dissect the contribution of proteins in additional molecular functions and also to reveal the aspects of protein and ncRNAs in several human diseases and malignancies.
Main objective of the present thesis was the study of the role of important RNA-binding protein subunits which participate in biogenesis of the tRNA molecules but could also additionally carry out alternative tasks in non-canonical functions. In the first part of the thesis, the role of La protein overexpression in cancer was examined as a global gatekeeper and abundance regulator of Pol III ncRNAs. La overexpression in lung adenocarcinoma cells lead to a significant increase in cell proliferation and motility. Next Generation sequencing (NGS) analysis showed the participation of La in translation and RNA metabolism. La accumulation affected the expression of mTORC2 complex and important tRNA maturation and translation initiation factors. In addition, upregulation of the tumorigenic tRF-1001 and its parent tRNASerUGA was also observed. Although the conserved LAM-RRM1 module is responsible for the RNA binding capacity of La, biochemical analysis showed that LAM motif alone can bind both oligo(U) and a pre-tRNA ligands. The biochemical, supported also by existing NMR-derived structural data, supports the La involvement in binding of diverse RNAs, that could account for a modulatory role of La in carcinogenesis.
In the second part of the thesis, the CRISPR/Cas9 genome editing tool was used to knock-out each individual protein subunit of RNase P holoenzyme in HeLa cancer cell line. Given the current knowledge on moonlighting roles of the 10 RBPs that form the RNase P holoenzyme in human cells, the screening analysis showed that POP1 and RPP30 protein subunits are essential for cell viability while RPP25 is dispensable. The RPP25 deficient cells showed an increased proliferation and migration rate. In a next step, Next Generation sequencing (NGS) analysis showed that ablation of RPP25 regulates genes with significant role in cancer and steroid metabolism but also it is observed a deregulation of expression levels of long-intergenic RNAs and other non-coding RNAs that participate in cancer. Moreover, small RNA-seq showed that specific miRNAs, tRNAs and tRFs are deregulated enhancing the cancer progression. Subsequent, global analysis of translation rates confirmed the elevated translational levels. Finally, the edited cell lines were used for in vivo characterization of RPP25-GFP tagged localization using fluorescence microscopy to reveal possible alternative localizations of RPP25 that could imply alternative functions in RNA-mediated phase separation.
Taken together, the current thesis attempted to provide a comprehensive picture of assembly, coordination, and heterogeneity of prominent RNA-binding proteins like La and RPP25, under various conditions and to identify pathways that regulate gene expression at the transcription and translation level, that could be further exploited for therapy.
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