Translational regulation is an important mechanism for regulating gene expression. Regulation of specific transcripts is often mediated by elements present in the 3′ untranslated region (UTR) of the mRNA. In the Drosophila embryo, translational repression of nanos mRNA is mediated by Smaug protein bound to specific sequences present in the 3′ UTR of the mRNA. Here I show that Smaug recruits Cup protein to the mRNA and that Cup in turn binds to the translation initiation factor eIF4E that is present at the 5′. The interaction between Cup and eIF4E prevents formation of a productive translation initiation complex on the mRNA. Therefore, Smaug-dependent translational repression functions at the initiation step via the indirect interaction of Smaug with eIF4E, which is mediated by Cup. In the second example of translational regulation presented here, I show that an element present in the 3′ untranslated region of the Hsp83 mRNA mediates translational enhancement in the Drosophila embryo. I have identified three proteins, Hrp48, DDP1 and PABP that interact with this element and have demonstrated that Hrp48 and DDP1 function in translational enhancement.

Translational regulation is an important mechanism for regulating gene expression. Regulation of specific transcripts is often mediated by elements present in the 3′ untranslated region (UTR) of the mRNA. In the Drosophila embryo, translational repression of nanos mRNA is mediated by Smaug protein bound to specific sequences present in the 3′ UTR of the mRNA. Here I show that Smaug recruits Cup protein to the mRNA and that Cup in turn binds to the translation initiation factor eIF4E that is present at the 5′. The interaction between Cup and eIF4E prevents formation of a productive translation initiation complex on the mRNA. Therefore, Smaug-dependent translational repression functions at the initiation step via the indirect interaction of Smaug with eIF4E, which is mediated by Cup. In the second example of translational regulation presented here, I show that an element present in the 3′ untranslated region of the Hsp83 mRNA mediates translational enhancement in the Drosophila embryo. I have identified three proteins, Hrp48, DDP1 and PABP that interact with this element and have demonstrated that Hrp48 and DDP1 function in translational enhancement.

Cytoplasmic polyadenylation-element binding protein (CPEB) is required for the translational regulation in multiple cell types. CPEB is known to play important roles in early germ cell development, in neuronal synaptic plasticity, and in the process of cellular senescence. CPEB is able to control translation by first interacting with a specific sequence of mRNA known as the CPE site. CPEB recognizes a specific sequence of mRNA, called the cytoplasmic polyadenylation element. This is a uracil rich sequence that is located on the 3' UTR of mRNA. Once CPEB is bound to the CPE site, CPEB can interact with other proteins. CPEB is most notably known for interacting with a cleavage and polyadenylation specificity factor (CPSF), with a poly(A)-specific ribonuclease, and with a poly(A) polymerase in the Gld2 family. This complex of proteins controls polyadenylation on the 3' end of mRNA. By controlling the lengthening of the poly(A) tail, translation can be regulated. CPEB is believed to contain two RNA recognition motifs and a zinc binding region on the N-terminus. The zinc binding region contains six cysteine and two histidine residues that bind to two zinc atoms in a tetrahedral geometry. Using NMR spectroscopy, the structure of zinc binding region of CPEB1 was determined. This protein was shown to bind to two zinc ions in a cross-braced topology. The zinc binding region of CPEB was also determined that the correct classification for this zinc finger is a ZZ domain.

Pumilio 2 (PUM2) is a member of the Puf family of mRNA binding proteins and translational regulators which are involved in various processes including embryonic patterning and memory formation. Nevertheless, its functions in the outgrowth of neuronal axons have not been studied. This study shows endogenous expression of PUM2 in neurites of dorsal root ganglia (DRG) neurons and transport of PUM2 along retinal ganglion cell (RGC) axons and their growth cones. Overexpression of PUM2 in DRG neurons resulted in shorter axons when compared to control neurons. Expression of either dominant negative mutation (dnPUM2) or PUM2W349G displayed a reduction in axonal length. PUM2 downregulation with microRNA (miRNA) also caused a reduction in neurite length compared to control neurons. Finally, PUM2 silencing did not alter eye size at E4, which allows investigation of axonal outgrowth in RGC in vivo. These results suggest a novel role for PUM2 in axonal outgrowth.

A biofilm is a complex community of cells enveloped in a self-produced polymeric matrix. Entry into a biofilm is exquisitely controlled at the level of transcription and in Bacillus subtilis it requires the concerted efforts of several major transcription factors including the repressor SinR and activator DegU. I initially identified that these transcriptional regulators control biofilm formation via parallel pathways. Through investigating the regulation of biofilm formation by SinR and DegU, I discovered that biofilm formation is also regulated at the post-translational level. This was achieved by identifying three key proteins which are needed for biofilm formation. These proteins are PtkA, a bacterial tyrosine kinase; TkmA, the cognate modulator of PtkA; and PtpZ, a bacterial tyrosine phosphatase. By introducing amino acid point mutations within the catalytic domains of PtkA and PtpZ it was identified that the kinase phosphatase activities, respectively, are essential function.In addition, PtkA contains a conserved C-tyrosine cluster that is the site autophosphorylation. Investigation of the role of the C-terminal tyrosine cluster tentatively suggests that this domain acts to block access to the active site of PtkA, thus affecting the ability of PtkA to phosphorylate its targets. Deletion of the gene coding for TkmA demonstrated that this modulator was also required for biofilm formation. It was also demonstrated that TkmA may interact with other protein partners, at least in the absence of PtkA, raising the question of how signal specificity is maintained. Finally, a systematic mutagenesis approach was used with the aim of identifying the target(s) of PtkA and PtpZ during biofilm formation but,despite extensive efforts, it remained elusive. The findings presented in this thesis highlight the complexity of biofilm formation by B. subtilis by revealing an additional level of regulation in the form of protein tyrosine phosphorylation.

Pumilio 2 (PUM2) is a member of the Puf family of mRNA binding proteins and translational regulators which are involved in various processes including embryonic patterning and memory formation. Nevertheless, its functions in the outgrowth of neuronal axons have not been studied. This study shows endogenous expression of PUM2 in neurites of dorsal root ganglia (DRG) neurons and transport of PUM2 along retinal ganglion cell (RGC) axons and their growth cones. Overexpression of PUM2 in DRG neurons resulted in shorter axons when compared to control neurons. Expression of either dominant negative mutation (dnPUM2) or PUM2W349G displayed a reduction in axonal length. PUM2 downregulation with microRNA (miRNA) also caused a reduction in neurite length compared to control neurons. Finally, PUM2 silencing did not alter eye size at E4, which allows investigation of axonal outgrowth in RGC in vivo. These results suggest a novel role for PUM2 in axonal outgrowth.

Early embryonic development in vertebrates is directed in part by maternal mRNAs expressed in oocytes and stored in cytoplasmic messenger ribonucleoprotein particles (mRNPs). Abundant evidence demonstrates the importance of mRNPs in embryonic development and in post-embryonic cellular function; however their characterization has been hampered by lack of suitable methodologies. The Xenopus oocyte has been the primary model system for studies of mRNPs. YY1 is a well-studied transcriptional regulatory factor that is sequestered in the oocyte cytoplasm and present entirely in cytoplasmic oocyte mRNPs. The objective of this thesis was to examine the biochemistry of YY1 association with maternal mRNA molecules in order to shed light on the role of YY1 in development and the poorly understood biology of oocyte mRNPs. The initial working hypotheses were that association of YY1 with mRNPs is dependent on sequence-specific RNA-binding activity and, therefore, that YY1 associates with a definite subset of maternal mRNA. A number of unique methods were developed in this study to address these hypotheses. RNA immunoprecipitation-DNA microarray (RIP-CHIP) analysis establishes that YY1 associates with a subset of mRNAs in the oocyte pool. A novel sequence-specific RNA-binding activity of the YY1 protein is demonstrated, and the RNA-binding activity of YY1 is shown to be required for its association with oocyte mRNPs in vivo. The functional roles of YY1 mRNA substrates are discussed in the context of embryological development and the biological function of YY1 in oocyte mRNPs. Extension of the experimental approaches developed in this thesis to the entire set of mRNP proteins would significantly advance our understanding of mRNP composition and heterogeneity, as well as the biological function of maternal mRNAs and mRNPs in development.

Early embryonic development in vertebrates is directed in part by maternal mRNAs expressed in oocytes and stored in cytoplasmic messenger ribonucleoprotein particles (mRNPs). Abundant evidence demonstrates the importance of mRNPs in embryonic development and in post-embryonic cellular function; however their characterization has been hampered by lack of suitable methodologies. The Xenopus oocyte has been the primary model system for studies of mRNPs. YY1 is a well-studied transcriptional regulatory factor that is sequestered in the oocyte cytoplasm and present entirely in cytoplasmic oocyte mRNPs. The objective of this thesis was to examine the biochemistry of YY1 association with maternal mRNA molecules in order to shed light on the role of YY1 in development and the poorly understood biology of oocyte mRNPs. The initial working hypotheses were that association of YY1 with mRNPs is dependent on sequence-specific RNA-binding activity and, therefore, that YY1 associates with a definite subset of maternal mRNA. A number of unique methods were developed in this study to address these hypotheses. RNA immunoprecipitation-DNA microarray (RIP-CHIP) analysis establishes that YY1 associates with a subset of mRNAs in the oocyte pool. A novel sequence-specific RNA-binding activity of the YY1 protein is demonstrated, and the RNA-binding activity of YY1 is shown to be required for its association with oocyte mRNPs in vivo. The functional roles of YY1 mRNA substrates are discussed in the context of embryological development and the biological function of YY1 in oocyte mRNPs. Extension of the experimental approaches developed in this thesis to the entire set of mRNP proteins would significantly advance our understanding of mRNP composition and heterogeneity, as well as the biological function of maternal mRNAs and mRNPs in development.

No description available.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第23818号 / 医科博第139号 / 新制||医科||9(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 萩原 正敏, 教授 藤渕 航, 教授 上杉 志成 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM