Post-graduate theses
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| Identifier |
000463896 |
| Title |
Τhe role of Rpb9 in non-coding regulation and cell fate decision-making in yeast |
| Alternative Title |
Ο ρόλος της Rpb9 στην μη κωδική ρύθμιση και στην επιλογή κυτταρικής μοίρας στην ζύμη |
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Author
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Μηνακάκης, Αλέξανδρος Β.
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Thesis advisor
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Moretto, Fabien
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Reviewer
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Σπηλιανάκης, Χαράλαμπος
Ντίνη, Ευγενία
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| Abstract |
Non-coding transcription is an extensive phenomenon in yeast, with multiple ncRNAs regulating the
expression of coding genes in different conditions and during cell-fate decision-making. Rpb9 is a nonessential RNA polymerase II subunit contributing to transcription start site selection, transcription elongation
and termination that has also been implicated in non-coding regulation, both in vegetative state (SER3) and
during entry into meiosis (IRT1). The aim of this master thesis was to study the contribution of Rpb9 in noncoding regulation and cell-fate decision-making in S.cerevisiae.
First, we showed that Rpb9 is required for proper regulation of the SER3 locus, as its absence leads to overexpression of SER3 but does not affect the transcription of the full-length SRG1 RNA, leading however to
increased production of SRG1 unstable transcripts. To further investigate the role of Rpb9 in non-coding
regulation in the SER3 locus and a genome-wide level we utilized transcriptomic and proteomic approaches.
Specifically, we implemented the nascent RNA CRAC protocol in Ctrl and Rpb9Δ haploid cells to study the
effect of Rpb9 in the dynamics of transcription. To investigate the possible interactions of RNAPII with other
factors Rpb9 might mediate we followed a proteomic approach and generated strains suitable for inducible
degradation of Rpb9 using the Auxin Inducible Degradation (AID) system and for interactomic assays.
We then focused our work in the role of Rpb9 in cell-fate decision-making and non-coding transcription during
meiosis. As Rpb9 has been identified as an activator of IRT1 (Moretto et al 2021) a non-coding RNA that
represses the master regulator of gametogenesis IME1, we decided to further expand upon its contribution in
the regulation of the IRT1/IRT2 circuit. Specifically, we proved that Rpb9 facilitates the expression of IRT2.
We also showed that the activation of IRT2 is not sufficient to induce IRT1 expression in the absence of Rpb9,
indicating that Rpb9 might act in both an IRT2-dependent and independent manner to regulate IRT1
expression.
We also expanded upon the importance of Rpb9 in the progression of gametogenesis by utilizing the AID
system to prove that Rpb9 has a distinct role in the progression of meiosis, as its degradation right before the
entry into gametogenesis is sufficient to delay the process and decrease the sporulation efficiency. We also
showed that Rpb9 is essential for proper chromosome segregation during meiosis as its absence leads to
increased miss-segregation. These results prompted us to investigate the role of Rpb9 in gene regulation during
gametogenesis. Specifically, we showed that Rpb9 is required for the regulation of NDC80, a gene that
encodes for a protein important in chromosome segregation, as its absence leads to loss of the Ndc80 pattern
during gametogenesis and delayed activation of NDC80LUTI, a ncRNA that represses NDC80ORF. We also
proved that Rpb9 is essential for the timely activation of other early-meiotic genes like IME2 and REC8.
Finally, to study in a genome-wide level the contribution of Rpb9 in meiosis-specific gene regulation we
followed a transcriptomic approach, by performing synchronous sporulation experiments and preparing RNA
samples for total, TSS and long-read RNA sequencing.
During this master thesis we have shown that Rpb9 is important for the regulation of three loci, regulated by
non-coding transcription with different mechanisms and in different conditions. We have also proved that
Rpb9 is important for cell-fate decision-making as it mediates the proper entry and progression of meiosis.
Finally, the transcriptomic and interactomic approaches we adapted could help us shed light upon the
mechanisms involved in Rpb9-mediated non-coding regulation.
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| Language |
English |
| Issue date |
2024-03-29 |
| Collection
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School/Department--School of Sciences and Engineering--Department of Biology--Post-graduate theses
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Type of Work--Post-graduate theses
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| Permanent Link |
https://elocus.lib.uoc.gr//dlib/4/8/9/metadata-dlib-1712134887-609809-11086.tkl
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| Views |
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