Experiments were performed to test sequence and structural specific interactions of proteins with a conserved RNA modification enzyme, which is known as Ime4 in yeast and Mettl3 in mammals. Ime4 methylates N6-adenosine bases on mRNA molecules. The goal of this project is to gain direct insights into how novel proteins interact with Ime4 to form the methyltranferase (MTase) complex and to identify proteins that are essential for Ime4 activity. It has been recognized that there are two proteins that interact within the Ime4 complex, which are known as Mum2 (a cytoplasmic protein essential for meiotic DNA replication within yeast) and Slz1 (a transcription factor). We hypothesize that the N-terminal domain of Ime4 is the location of binding of the aforementioned proteins in this complex. Similarly, we tested whether the human ortholog of Ime4 (Mettl3) forms an analogous complex that includes an ortholog of Mum2, known as WTAP, and its binding partner WT1. The major approaches include in vivo genetic assays in yeast to test protein-protein interactions and the use of recombinant DNA technology to construct fusion genes/deletions. The results demonstrate that Mum2 interacts with a specific, non-conserved region in the Ime4 N-terminal domain. Furthermore, we discovered a new binding partner, Ygl036w, which also interacts with Ime4. Currently, several experiments are being carried out with the Mettl3 complex and its hypothesized protein binding partners to assess the interactions of this complex.

京都大学 / 新制・課程博士 / 博士(薬学) / 甲第23148号 / 薬博第848号 / 新制||薬||242(附属図書館) / 京都大学大学院薬学研究科薬学専攻 / (主査)教授 土居 雅夫, 教授 中山 和久, 教授 竹島 浩 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(薬科学) / 甲第22046号 / 薬科博第112号 / 新制||薬科||12(附属図書館) / 京都大学大学院薬学研究科医薬創成情報科学専攻 / (主査)教授 土居 雅夫, 准教授 Fustin,Jean Michel, 教授 中山 和久 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

RNA modifications and processing events are important modulators of global gene expression. Genomic mutations in the RNA methylase NSun2 and the alternative splicing factor Srsf2 are linked to neurological disorders and cancer in humans, respectively. NSun2 methylates cytosine-5 in most tRNAs and, to a lesser extent, other ncRNAs and mRNAs. Srsf2 is a critical component of the spliceosome and interacts with abundant ncRNAs that are methylated by NSun2. However, how precisely these processes effect homeostasis is largely unexplored. Therefore, the main aims of my PhD were (1) to dissect the molecular mechanisms of NSun2-mediated RNA methylation pathways that regulate cell survival under normal conditions and in response to oxidative stress, and (2) to investigate the importance of Srsf2 in stem cells using skin as a model system. In the context of RNA modifications, firstly I described how NSun2-expressing cells enrich for transcripts related to enhanced cell survival. Subsequently, by metabolically profiling wildtype and patient-derived dermal fibroblasts carrying loss-of-function mutations in the NSUN2 gene, I showed that the absence of NSun2 is synonymous to an energy-saving, low-translating and stressed cellular state. I further confirmed that lack of NSun2 was sufficient to instigate a cellular stress response, by monitoring BIRC5, a member of the inhibitor of apoptosis family. To further answer whether lack of NSun2 enhanced the susceptibility of patient cells to external stress stimuli, I next exposed them to oxidative stress and measured transcriptional and translational changes. I discovered that NSun2 is required to adapt global protein synthesis to the stress response, while NSun2-depleted cells failed to do so. This was concurrent with NSun2-depleted cells enriching for transcripts related to mRNA degradation and negative regulators of protein translation in response to stress. Generally, since loss of NSun2-driven methylation in tRNAs triggers their cleavage into small ncRNA fragments by angiogenin, I asked how angiogenin or tRNA-derived ncRNAs affect translation levels. In the presence of NSun2, angiogenin alone did not reduce global protein synthesis, yet tRNA fragmentation was required to modulate translation levels. Finally, to uncover how the lack of NSun2 influenced tRNA cleavage and methylation patterns in response to stress, I exposed wildtype and patient cells to sodium arsenite and measured the abundance of tRNA-derived fragments and occurrence of methylation events. With this I discovered unique tRNA fragmentation patterns and global RNA methylation profiles for wildtype and NSun2-depleted cells, that can account for the underlying molecular and phenotypical differences in response to stress. In the context of alternative splicing, and since the cellular functions of Srsf2 are largely unknown, I explored its role in cellular survival and differentiation. By conditionally deleting SRSF2 in two different stem cell populations of the mouse epidermis, I observed significant thickening of the epidermis, altered expression of cell proliferation and stem cell differentiation markers, and distorted hair follicle structures. Moreover, I demonstrated that lack of Srsf2 promotes skin regeneration following injury, thus strongly indicating that Srsf2 is required for normal skin development and regeneration after injury. In summary, my research suggests that NSun2-mediated RNA methylation pathways orchestrate transcriptional and translational programmes in response to external stress stimuli, and my studies are the first to show that the alternative splicing factor Srsf2 is required for stem cell differentiation in skin.

Argonaute proteins are small DNA/RNA-guided endonucleases found in all domains of life. In animals, small RNAs of length 21–35 nucleotides direct the PIWI-clade of Argonautes to silence complementary target RNAs; these are called PIWI-interacting RNAs (piRNAs). During spermatogenesis in mice, piRNA-guided PIWI proteins, MIWI2, MILI, and MIWI, silence transposons, regulate expression of protein-coding genes and are necessary for fertility. A working endonuclease activity of MIWI and MILI is essential to complete spermatogenesis. Yet, both MIWI and MILI produce weak and slow target cleavage in vitro, thwarting biochemical examination of the silencing step. Here, we find that PIWI proteins require an auxiliary protein to efficiently cleave their targets, unlike any other known Argonaute. Gametocyte Specific Factor 1 (GTSF1) is a conserved zinc-finger protein essential for fertility and piRNA-directed silencing. We show GTSF1 accelerates the pre-steady-state rate of target cleavage by MIWI and MILI; this role of GTSF1 is also preserved in insects. A critical step in GTSF1 mechanism entails binding RNA. GTSF1 allowed detailed kinetic analyses of catalytic PIWIs: they require extensive 3′ complementarity between the guide and target to efficiently cleave them, but this base-pairing also limits turnover. Interestingly, within a species, different PIWI proteins have unique kinetic properties. In sum, our findings provide molecular mechanisms of GTSF1 function and target silencing by PIWIs as well as a useful method for future studies.

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