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<p>Gene expression in the eukaryotic cell is a fundamental cellular process, which consists of several distinct steps but extensively coupled to each other. From site of transcription in the nucleus to the cytoplasm, both mRNA and rRNA are associated with a proper set of proteins. These proteins influence RNA processing, transport as well as ribosome maturation. We have tried to take advantage of different model systems to understand the process of eukaryotic gene expression at the post-transcription level. To this end, we have focused on identification and characterization of several specific proteins in the context of mRNP and rRNP particles.</p><p>We have characterized a novel yeast gene MRD1, which encodes a protein with five RNA-binding domains (RBDs) and is essential for viability. Mrd1p is present in the nucleolus and the nucleoplasm. Depletion of Mrd1p leads to a decrease in the synthesis of 18S rRNA and 40S ribosomal subunits. Mrd1p associates with the 35S prerRNA and the U3 snoRNA and is required for the initial processing of pre-rRNA at the A₀-A₂ sites. The presence of five RBDs in Mrd1p suggests that Mrd1p may function to correctly fold pre-rRNA, a requisite for proper cleavage.</p><p>Meanwhile, an MRD1 homologue, Ct-RBD-1 with six RBDs, has also been identified and shown to involve in ribosome biogenesis in Chironomus tentans. Ct-RBD-1 binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In the cytoplasm, Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that Ct-RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes.</p><p>We have characterized a novel abundant nucleolar protein, p100 in C. tentans. The p100 protein is located in the fibrillar compartment of the nucleolus, and remains in the nucleolus after digestion with nucleases. This indicates that p100 might be a constituent of the nucleolar proteinaceous framework. Remarkably, p100 is also localized in the brush border in the apical part of the salivary gland cell. These results suggest that it could be involved in coordination of the level of protein production and export from the cell through regulation of the level of rRNA production in the nucleolus.</p><p>We have characterized a Dbp5 homologue in C. tentans, Ct-Dbp5. The protein becomes associated with nascent pre-mRNAs at a large number of active genes, including the Balbiani ring (BR) genes. Ct-Dbp5 is bound to nascent BR pre-mRNP particles and accompanies them through the nucleoplasm and the nuclear pore into the cytoplasm. Nuclear accumulation of Ct-Dbp5 takes place when synthesis and/or export of mRNA are inhibited. Our results indicate that most or all of the shuttling Ct-Dbp5 exiting from the nucleus associated with mRNP. Furthermore, Ct-Dbp5 is present along the mRNP fibril extending into the cytoplasm, supporting the view that Ct-Dbp5 is involved in restructuring the mRNP prior to translation.</p><p>We have shown that the export receptor CRM1 in C. tentans is associated with BR pre-mRNP while transcription takes place. We have also shown that the GTPase Ran binds to BR pre-mRNP, but its binding mainly in the interchromatin. Although both CRM1 and Ran accompany BR pre-mRNP through the nuclear pore, Leptomycin B treatment reveals that a NES-CRM1-RanGTP complex is not essential for export of the BR mRNP. Our results suggest that several export receptors associate with BR mRNP and that these receptors might have redundant functions in the nuclear export of BR mRNP.</p><p>We have analyzed four SR proteins, SC35, ASF/SF2, 9G8 and hrp45, in C. tentans. All four SR proteins genes are expressed in salivary gland cells and in several other tissues in a tissue specific pattern. We found that about 90% of all nascent pre-mRNAs bind all four SR proteins, and that approximately 10% of the pre-mRNAs associate with different subsets of the four SR proteins, suggesting that not all of four SR proteins are needed for processing of pre-mRNA. None of three examined SR proteins leave BR pre-mRNP as splicing is completed. Instead, 9G8 accompanies the mRNP to the cytoplasm, while SC35 and hrp45 leave the BR mRNP at the nuclear side of the nuclear pore complex.</p>

Gene expression in the eukaryotic cell is a fundamental cellular process, which consists of several distinct steps but extensively coupled to each other. From site of transcription in the nucleus to the cytoplasm, both mRNA and rRNA are associated with a proper set of proteins. These proteins influence RNA processing, transport as well as ribosome maturation. We have tried to take advantage of different model systems to understand the process of eukaryotic gene expression at the post-transcription level. To this end, we have focused on identification and characterization of several specific proteins in the context of mRNP and rRNP particles. We have characterized a novel yeast gene MRD1, which encodes a protein with five RNA-binding domains (RBDs) and is essential for viability. Mrd1p is present in the nucleolus and the nucleoplasm. Depletion of Mrd1p leads to a decrease in the synthesis of 18S rRNA and 40S ribosomal subunits. Mrd1p associates with the 35S prerRNA and the U3 snoRNA and is required for the initial processing of pre-rRNA at the A0-A2 sites. The presence of five RBDs in Mrd1p suggests that Mrd1p may function to correctly fold pre-rRNA, a requisite for proper cleavage. Meanwhile, an MRD1 homologue, Ct-RBD-1 with six RBDs, has also been identified and shown to involve in ribosome biogenesis in Chironomus tentans. Ct-RBD-1 binds pre-rRNA in vitro and anti-Ct-RBD-1 antibodies repress pre-rRNA processing in vivo. Ct-RBD-1 is mainly located in the nucleolus in an RNA polymerase I transcription-dependent manner, but it is also present in discrete foci in the interchromatin and in the cytoplasm. In the cytoplasm, Ct-RBD-1 is associated with ribosomes and, preferentially, with the 40S ribosomal subunit. Our data suggest that Ct-RBD-1 plays a role in structurally coordinating pre-rRNA during ribosome biogenesis and that this function is conserved in all eukaryotes. We have characterized a novel abundant nucleolar protein, p100 in C. tentans. The p100 protein is located in the fibrillar compartment of the nucleolus, and remains in the nucleolus after digestion with nucleases. This indicates that p100 might be a constituent of the nucleolar proteinaceous framework. Remarkably, p100 is also localized in the brush border in the apical part of the salivary gland cell. These results suggest that it could be involved in coordination of the level of protein production and export from the cell through regulation of the level of rRNA production in the nucleolus. We have characterized a Dbp5 homologue in C. tentans, Ct-Dbp5. The protein becomes associated with nascent pre-mRNAs at a large number of active genes, including the Balbiani ring (BR) genes. Ct-Dbp5 is bound to nascent BR pre-mRNP particles and accompanies them through the nucleoplasm and the nuclear pore into the cytoplasm. Nuclear accumulation of Ct-Dbp5 takes place when synthesis and/or export of mRNA are inhibited. Our results indicate that most or all of the shuttling Ct-Dbp5 exiting from the nucleus associated with mRNP. Furthermore, Ct-Dbp5 is present along the mRNP fibril extending into the cytoplasm, supporting the view that Ct-Dbp5 is involved in restructuring the mRNP prior to translation. We have shown that the export receptor CRM1 in C. tentans is associated with BR pre-mRNP while transcription takes place. We have also shown that the GTPase Ran binds to BR pre-mRNP, but its binding mainly in the interchromatin. Although both CRM1 and Ran accompany BR pre-mRNP through the nuclear pore, Leptomycin B treatment reveals that a NES-CRM1-RanGTP complex is not essential for export of the BR mRNP. Our results suggest that several export receptors associate with BR mRNP and that these receptors might have redundant functions in the nuclear export of BR mRNP. We have analyzed four SR proteins, SC35, ASF/SF2, 9G8 and hrp45, in C. tentans. All four SR proteins genes are expressed in salivary gland cells and in several other tissues in a tissue specific pattern. We found that about 90% of all nascent pre-mRNAs bind all four SR proteins, and that approximately 10% of the pre-mRNAs associate with different subsets of the four SR proteins, suggesting that not all of four SR proteins are needed for processing of pre-mRNA. None of three examined SR proteins leave BR pre-mRNP as splicing is completed. Instead, 9G8 accompanies the mRNP to the cytoplasm, while SC35 and hrp45 leave the BR mRNP at the nuclear side of the nuclear pore complex.

Bcl-2 associated factor 1 (Bclaf1; previously known as Btf) is a nuclear protein that was originally identified as an interacting partner for the adenoviral anti-apoptotic Bcl-2 family member E1B-19K. Surprisingly, Bclaf1 does not share structural homology with the Bcl-2 family of proteins, but rather exhibits protein structure and subcellular distribution patterns reminiscent of proteins that regulate mRNA processing. In addition, Bclaf1 appears to be expressed at high levels in skeletal muscle and was recently shown to associate with emerin, a protein linked to muscular dystrophy. Despite these observations, roles for Bclaf1 in RNA processing and/or skeletal muscle differentiation remain to be elucidated. In an effort to identify new roles for Bclaf1 I conducted protein-protein interaction screens to identify candidate interacting proteins and pathways. I identified p32 and 9G8 as novel interacting partners for Bclaf1. Additional subsequent experiments demonstrated an interaction of Bclaf1 with tip associated protein (Tap) and association of Bclaf1 with ribonucleoprotein complexes. Given that all of these proteins have been linked to mRNA processing, a role for Bclaf1 in this pathway was investigated. Using several approaches, I demonstrated that Bclaf1 is able to associate with splicing complexes and mRNA species at various stages of processing. The function of Bclaf1 in the context of skeletal muscle differentiation was also explored using skeletal muscle cell lines and primary mouse myoblasts. Skeletal muscle differentiation led to a dramatic decrease in nuclear Bclaf1 steady-state protein, with the unexpected appearance of smaller Bclaf1 protein species that accumulated in the cytoplasm during differentiation due to cleavage by caspases. Furthermore, Bclaf1 depletion in a myoblast cell line led to increased myoblast fusion and myofiber dimensions during differentiation. Overall our findings indicate roles for Bclaf1 in the skeletal muscle differentiation program and in molecular events that regulate pre-mRNA splicing and related events.

Bcl-2 associated factor 1 (Bclaf1; previously known as Btf) is a nuclear protein that was originally identified as an interacting partner for the adenoviral anti-apoptotic Bcl-2 family member E1B-19K. Surprisingly, Bclaf1 does not share structural homology with the Bcl-2 family of proteins, but rather exhibits protein structure and subcellular distribution patterns reminiscent of proteins that regulate mRNA processing. In addition, Bclaf1 appears to be expressed at high levels in skeletal muscle and was recently shown to associate with emerin, a protein linked to muscular dystrophy. Despite these observations, roles for Bclaf1 in RNA processing and/or skeletal muscle differentiation remain to be elucidated. In an effort to identify new roles for Bclaf1 I conducted protein-protein interaction screens to identify candidate interacting proteins and pathways. I identified p32 and 9G8 as novel interacting partners for Bclaf1. Additional subsequent experiments demonstrated an interaction of Bclaf1 with tip associated protein (Tap) and association of Bclaf1 with ribonucleoprotein complexes. Given that all of these proteins have been linked to mRNA processing, a role for Bclaf1 in this pathway was investigated. Using several approaches, I demonstrated that Bclaf1 is able to associate with splicing complexes and mRNA species at various stages of processing. The function of Bclaf1 in the context of skeletal muscle differentiation was also explored using skeletal muscle cell lines and primary mouse myoblasts. Skeletal muscle differentiation led to a dramatic decrease in nuclear Bclaf1 steady-state protein, with the unexpected appearance of smaller Bclaf1 protein species that accumulated in the cytoplasm during differentiation due to cleavage by caspases. Furthermore, Bclaf1 depletion in a myoblast cell line led to increased myoblast fusion and myofiber dimensions during differentiation. Overall our findings indicate roles for Bclaf1 in the skeletal muscle differentiation program and in molecular events that regulate pre-mRNA splicing and related events.

京都大学 / 新制・課程博士 / 博士(理学) / 甲第24082号 / 理博第4849号 / 新制||理||1694(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)准教授 柘植 知彦, 教授 森 和俊, 教授 川口 真也 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM