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The regulation of cardiac-specific genes such as GATA-4 and its co-factor FOG-2 is paramount for normal heart development and function. Indeed, those mechanisms that regulate GATA-4 and FOG-2 function, such as nuclear transport and the post-translational modification of SUMOylation, are of critical importance for cardiogenesis. Therefore the aims of this study were to: i) elucidate the nuclear transport mechanisms of GATA-4; ii) determine the function of SUMOylation on the biological activity of both GATA-4 and FOG-2; and iii) examine how these mechanisms impact on the role of GATA-4 and FOG-2 in cardiac hypertrophy. Firstly, we characterised a non-classical nuclear localisation signal that mediates active import of GATA-4 in both HeLa cells and cardiac myocytes. Fine mapping studies revealed four crucial residues within this region that interacted with importin ?? to mediate GATA-4 import via the non-classical import pathway. In addition, a cardiac myocyte-specific CRM1-dependent nuclear export signal, which consists of three essential leucine residues, was identified. We also investigated the residues of GATA-4 that are responsible for its DNAbinding activity and therefore transcriptional control of cardiac-specific genes. Secondly, we demonstrated that SUMOylation of both GATA-4 and FOG-2 is exclusively carried out by SUMO-2/3. Moreover, SUMOylation is involved in the nuclear localisation of both GATA-4 and FOG-2 in cardiac myocytes as well as the transcriptional regulation of cardiac-specific genes, such as cardiac troponin I. Finally, and perhaps most biologically significant, we showed that nuclear transport as well as SUMOylation of GATA-4 is imperative for the ability of GATA-4 to induce cardiac hypertrophy. Moreover, it was determined that FOG-2 SUMOylation is involved in the ability of FOG-2 to protect against cardiac hypertrophy. In conclusion, the current study provides detailed information on the nuclear transport pathways of GATA-4 as well as the SUMOylation of both GATA-4 and FOG-2 and the role these two mechanisms play in gene transcription and cardiac hypertrophy.

A synthetic pathway towards the cap-structure of 2,2,7-trimethylguanosine containing a methylene modified triphosphate bridge have been investigated. The modification to the triphosphate bridge is hoped to slow down cap degradation and give the connected  oligunucleotide an increased lifetime. This could result in an better understanding of nuclear transport of oligonucleotides and could thereby helping to develop new treatments for different diseases. The synthesis relies on a coupling reaction between the 2,2,7-trimethylguanosine 5’phosphate and 2’-O-methyladenosine with a 5’-pyrophosphate where the central oxygen has been replaced by a methylene group. The reaction pathway consists of 9 steps of which 8 steps have been successfully performed. The last step, which includes a coupling reaction, was attempted but without successful identification and isolation of the cap-structure, and will need further attention. The reaction has been performed in a milligram scale with various yields. / Presentation utförd

Doctor of Philosophy / Department of Diagnostic Medicine/Pathobiology / Raymond R. Rowland / Severe acute respiratory syndrome (SARS) is caused by an enveloped, positive-stranded RNA virus, the SARS coronavirus (SARS-CoV). Coronaviruses along with the arteriviruses are placed in the order, Nidovirales. Even though nidovirus replication is restricted to the cytoplasm, the nucleocapsid protein (N) of several coronaviruses and arteriviruses, localize to the nucleolus during infection. Confocal microscopy of N protein localization in Vero cells infected with the SARS-CoV or transfected with the SARS-CoV N gene failed to show presence of N in the nucleoplasm or nucleolus. Recombinant N remained cytoplasmic after the addition of leptomycin B (LMB), a drug that inhibits nuclear export. SARS-CoV N possesses a unique lysine-rich domain, located between amino acids 369-389, which possesses several nuclear localization signal (NLS) and nucleolar localization signal (NoLS) motifs. A chimeric protein composed of the 369-389 peptide substituted for the NLS of equine infectious anemia virus (EIAV) Rev protein (ERev) showed no nuclear localization activity. Three negatively charged amino acids, located at positions 372, 377 and 379 in SARS-CoV N were hypothesized to play a role in the loss of nuclear targeting. Substitution of aspartic acid-372 with alanine restored nuclear localization to the chimeric protein. A full-length recombinant SARS-N protein with the alanine-372 substitution localized to the nucleus. Therefore, the presence of an aspartic acid at position 372 is sufficient to retain N in the cytoplasm The mechanistic basis for how aspartic acid-372 interrupts nuclear transport is unknown, but may lie in the electrostatic repulsion with negatively charged amino acids located within the NLS binding pocket of importin-alpha.

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京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第23333号 / 生博第451号 / 新制||生||60(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 永尾 雅哉, 教授 片山 高嶺, 教授 谷口 雄一 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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Authors are permitted to post the MBoC PDF of their articles (and/or supplemental material) on their personal websites or in an online institutional repository provided there appears always the proper citation of the manuscript in MBoC and a link to the original publication of the manuscript in MBoC (http://www.molbiolcell.org/site/misc/ifora.xhtml) / 京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第19869号 / 生博第350号 / 新制||生||46(附属図書館) / 32905 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 河内 孝之, 教授 藤田 尚志, 教授 永尾 雅哉 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

The nuclear pore complex (NPC) is a proteinaceous gateway embedded in the nuclear envelope (NE) that regulates nucleocytoplasmic transport of molecules in eukaryotes. The NPC is formed by hundreds of proteins that are classified into approximately thirty different types of proteins called nucleoporin (Nup), each presents in multiples of eight copies. These nucleoporins are divided into two categories: the scaffold Nups forming the main structure of the NPC and the phenylalanine-glycine (FG) Nups that contain multiple repeats of intrinsically disordered and hydrophobic FG domains. These FG-Nups constitute the selective permeability barrier in the central channel of the NPC, which mediates the nuclear import of proteins into the nucleus, and the nuclear export of mRNA and pre-ribosomal subunits out of the nucleus. However, the precise copies of these Nups and their specific roles in the nucleocytoplasmic transport mechanism remain largely unknown. Moreover, the dysfunctional nuclear transport and the mutations of Nups have been closely associated with numerous human diseases, such as cancer, tumor and liver cirrhosis. We have developed and employed live-cell high-speed single-molecule microscopy to elucidate these critical questions remained in the nuclear transport and provide the fundamental knowledge for developing therapies. In this dissertation, I will present my major findings for the following three research projects: 1) determine the dynamic components of FG-Nups in native NPCs; 2) track the nucleocytoplasmic transport of transcription factor Smad proteins under ligand-activated conditions; and 3) elucidate the relationship between the nuclear export of mRNA and the presence and absence of specific Nups in live cells.Determination of the dynamic components for FG-Nups in native NPCs. Scaffold Nups have been intensively studied with electron microscopy to reveal their spatial positions and architecture in the past decades. However, the spatial organization of FG-Nups remains obscure due to the challenge of probing these disordered and dynamic polypeptides in live NPCs. By employing high-speed single-molecule microscopy and a live cell HaloTag labeling technique, I have mapped the spatial distribution for all eleven known mammalian FG-Nups within individual NPCs. Results show that all FG-Nups within NPCs are distinct in conformations and organized to form a ~300nm long hourglass shaped toroidal channel through the nuclear envelope. Exceptionally, the two remaining Nups (Nup98 and hCG1) almost extend through the entire NPC and largely overlap with all other FG-Nups in their spatial distributions. These results provide a complete map of FG-Nup organization within the NPC and also offer structural and functional insights into nucleocytoplasmic transport models. Tracking of the nucleocytoplasmic transport of Smad proteins under ligand-activated conditions. The inducement of transforming growth factor β1 (TGF-β1) was reported to cause the nuclear accumulation of Smad2/Smad4 heterocomplexes. However, the relationship between nuclear accumulation and the nucleocytoplasmic transport kinetics of Smad proteins in the presence of TGF-β1 remains obscure. By combining a high-speed single-molecule tracking microscopy technique (FRET), I tracked the entire TGF-β1-induced process of Smad2/Smad4 heterocomplex formation, as well as their transport through nuclear pore complex in live cells. The FRET results have revealed that in TGF-β1-treated cells, Smad2/Smad4 heterocomplexes formed in the cytoplasm, imported through the nuclear pore complexes as entireties, and finally dissociated in the nucleus. Moreover, it was found that basal-state Smad2 or Smad4 cannot accumulate in the nucleus without the presence of TGF-β1, mainly because both of them have an approximately twofold higher nuclear export efficiency compared to their nuclear import. Remarkably and reversely, heterocomplexes of Smad2/Smad4 induced by TGF-β1 can rapidly concentrate in the nucleus because of their almost fourfold higher nuclear import rate in comparison with their nuclear export rate. Thus, these single-molecule tracking data elucidate the basic molecular mechanism to understand nuclear transport and accumulation of Smad protein. Elucidation of the relationship between the nuclear export of mRNA and the presence and absence of specific Nups in live cells. In addition to explore the dynamic organization of NPC, in vivo characterization of the exact copy number and the specific function of each nucleoporin in the nuclear pore complex (NPC) remains desirable and challenging. Using live-cell high-speed super-resolution single-molecule microscopy, we first quantify the native copies of nuclear basket FG-Nups (Nup153, Nup50 and Tpr). Second, with same imaging technique and the auxin-inducible degradation strategies, I track the nuclear export of mRNA through native NPCs in absence of these FG-Nups. I found that these FG-Nups proteins possess the stoichiometric ratio of 1:1:1 and play distinct roles in the nuclear export of mRNAs in live cells. Tpr’s absence in the NPC dominantly reduces nuclear mRNA’s probability of entering the NPC for export. Complete depletion of Nup153 causes mRNA’s successful nuclear export efficiency dropped approximately four folds. Remarkably, the relationship between mRNA’s successful export efficiency and the copy number of Nup153 is not linear but instead follows a sigmoid function, in which mRNA can gain its maximum successful export efficiency as Nup153 increased from zero to around half of their full copies in the NPC. Lastly, the absence of Tpr or Nup153 also alters mRNA’s export routes through the NPC, but the removal of only Nup50 has almost no impact upon mRNA export route and kinetics. / Biology