Kaiso is a POZ-ZF transcription factor initially identified as an interaction partner for the cell adhesion co-factor p120^ctn. Kaiso-DNA binding is inhibited by p120^ctn, implicating p120^ctn in the regulation of Kaiso transcriptional activity. In this study, Kaiso repressed transcription of a luciferase reporter carrying four copies of the sequence-specific Kaiso-binding site (4xKBS) in artificial promoter assays. Mutation of the 4xKBS which is known to disrupt Kaiso-DNA binding also abrogated Kaiso-mediated transcriptional repression. Moreover, p120^ctn inhibited Kaiso-mediated transcriptional repression via the 4xKBS, yet neither the p120^ctn deletion mutant ΔR3-ll (lacking the Kaiso binding site) or p120^ctn NLS mutant (which cannot enter the nucleus) inhibited transcriptional repression. Furthermore, in NIH 3T3 cells (which do not demonstrate a Kaiso-pl20ctn interaction), pl20ctn failed to inhibit transcriptional repression. Many POZZF transcriptional repressors recruit an HDAC complex via their POZ domain to repress transcription. To investigate the mechanism of Kaiso-mediated transcriptional repression, the POZ domain of Kaiso was deleted, which abrogated transcriptional repression. Kaiso immunoprecipitates contained HDAC activity, and the HDAC co-repressor Sin3A co-immunoprecipitated with Kaiso, implying that Kaiso recruits Sin3A to repress transcription in an HDAC-dependent manner. Lastly, Kaiso repressed transcription via a human 𝑚𝑎𝑡𝑟𝑖𝑙𝑦𝑠𝑖𝑛 promoter fragment. This suggests that the KBS element is functionally relevant and implicates 𝑚𝑎𝑡𝑟𝑖𝑙𝑦𝑠𝑖𝑛 as a Kaiso target-gene. Collectively, these data establish Kaiso as a sequence-specific, HDAC-dependent transcriptional repressor that is regulated by the adhesion co-factor p120^ctn. / Thesis / Master of Science (MSc)

No description available.

Retroelements are genomic parasites which make up ~42% of the human genome and 38% of the mouse genome. Most are degenerate, but a large number have relatively intact promoter elements, suggesting that they are capable of transcription. Transcriptionally active retroelements can perturb normal transcription units in their vicinity through a variety of mechanisms, leading to phenotypic effects and in some cases disease. This phenomenon of transcriptional interference has been observed in organisms as diverse as maize, Drosophila, and the mouse. We analysed the extent of retroelement transcription in normal and diseased tissues, by searching the mouse and human EST databases for transcripts originating in retroelement promoters, and found a large number of transcripts from LINEs, SINEs and ERVs. Retroelement transcripts were found to be initiated in both sense and antisense orientations, and to be equally as common in normal and diseased tissue. Several of these transcripts were chimeric, appearing to initiate in retroelements and reading through to cellular genes, suggestive of transcriptional interference. We have used transposon display to identify and recover retroelement transcripts in the mouse. Transcripts initiated in LINE, SINE and ERV promoters are numerous, and many are chimeric with cellular genes. Although the numbers of recovered chimeric transcripts are too large to permit rigorous analysis of more than a small proportion, some of those we have studied further appear to be authentic transcripts that may represent interference with the canonical promoters of the genes in question. Our results suggest that transcriptional interference by retroelements may be a relatively common occurrence in mammals.

Ro ribonucleoproteins are antigenic protein-RNA particles that are the major targets of the immune reaction in autoimmune disorders like systemic lupus erythematosus. The Ro protein has been implicated in cellular RNA quality control, due to its preference for binding misfolded non-coding RNAs such as pre5S ribosomal RNAs and U2 small-nuclear RNAs besides binding cytoplasmic RNAs called Y RNAs. Although well characterized in eukaryotes, an understanding of Ro in prokaryotes is lacking. To gain structural insight into Ro-RNA interactions we have determined a high resolution crystal structure of Rsr, a Ro ortholog from the bacterium D. radiodurans. The structure of Rsr reveals two domains- a flexible, RNA binding HEAT repeat domain and a cation binding vonWillebrand factor A domain. Structural differences between Rsr and Xenopus laevis Ro at the misfolded non-coding RNA binding site suggest a possible conformational switch in Ro that might enable RNA binding. Structural and biochemical characterization reveals that Ro binds cytoplasmic small RNAs called Y RNAs with low nanomolar affinity, to form ~700kDa multimers. Formation of these multimers suggests one possible mode by which Ro RNAs may be targeted towards downstream processing events. Metal responsive transcriptional regulators sense specific metals in the cells and regulate the expression of specific operons involved in export, import or sequestration of the metal. CsoR is a copper(I) specific transcriptional regulator of the cso operon which consists of a putative copper export pump, CtpV. In copper limiting conditions, CsoR binds the operator/promoter region of the cso operon. In increased concentrations of copper (I), CsoR binds copper (I) with high affinity and is released from the operator/promoter site, causing derepression of the cso operon. To gain structural insight into CsoR function, we have solved the crystal structure of copper(I) bound CsoR. The structure reveals a homodimer with a subunit bridging copper site. The trigonal planar geometry and the presence of cysteine and histidine ligands at the metal site are favorable for copper(I) binding. The structure reveals a novel DNA binding fold in CsoR, making it the founding member of a new structural class of metalloregulators.

Vts1p is the Saccharomyces cerevisiae member of the Smaug family of post-transcriptional regulators, which is a group of sequence-specific RNA-binding proteins that regulate target mRNA expression. Vts1p is known to mediate deadenylation-dependent degradation of target transcripts through the recruitment of the Ccr4p/Pop2p/Not deadenylase complex. By conducting a functional analysis of Vts1p deletion mutants, I demonstrate that two regions within Vts1p are independently capable of downregulating the expression of an mRNA reporter. I provide both genetic and biochemical evidence that suggests residues 170-523 regulate reporter expression at the level of mRNA stability and function through a mechanism that requires the Ccr4p/Pop2p/Not deadenylase, whereas residues 1-237 repress reporter expression at the level of translation and function through a novel mechanism. In addition, I map a direct interaction between the eIF4E-binding protein, Eap1p, and the Vts1p SAM domain, which suggests a model in which residues 170-523 recruit Eap1p to mediate efficient target transcript degradation.

Vts1p is the Saccharomyces cerevisiae member of the Smaug family of post-transcriptional regulators, which is a group of sequence-specific RNA-binding proteins that regulate target mRNA expression. Vts1p is known to mediate deadenylation-dependent degradation of target transcripts through the recruitment of the Ccr4p/Pop2p/Not deadenylase complex. By conducting a functional analysis of Vts1p deletion mutants, I demonstrate that two regions within Vts1p are independently capable of downregulating the expression of an mRNA reporter. I provide both genetic and biochemical evidence that suggests residues 170-523 regulate reporter expression at the level of mRNA stability and function through a mechanism that requires the Ccr4p/Pop2p/Not deadenylase, whereas residues 1-237 repress reporter expression at the level of translation and function through a novel mechanism. In addition, I map a direct interaction between the eIF4E-binding protein, Eap1p, and the Vts1p SAM domain, which suggests a model in which residues 170-523 recruit Eap1p to mediate efficient target transcript degradation.

Ro ribonucleoproteins are antigenic protein-RNA particles that are the major targets of the immune reaction in autoimmune disorders like systemic lupus erythematosus. The Ro protein has been implicated in cellular RNA quality control, due to its preference for binding misfolded non-coding RNAs such as pre5S ribosomal RNAs and U2 small-nuclear RNAs besides binding cytoplasmic RNAs called Y RNAs. Although well characterized in eukaryotes, an understanding of Ro in prokaryotes is lacking. To gain structural insight into Ro-RNA interactions we have determined a high resolution crystal structure of Rsr, a Ro ortholog from the bacterium D. radiodurans. The structure of Rsr reveals two domains- a flexible, RNA binding HEAT repeat domain and a cation binding vonWillebrand factor A domain. Structural differences between Rsr and Xenopus laevis Ro at the misfolded non-coding RNA binding site suggest a possible conformational switch in Ro that might enable RNA binding. Structural and biochemical characterization reveals that Ro binds cytoplasmic small RNAs called Y RNAs with low nanomolar affinity, to form ~700kDa multimers. Formation of these multimers suggests one possible mode by which Ro RNAs may be targeted towards downstream processing events. Metal responsive transcriptional regulators sense specific metals in the cells and regulate the expression of specific operons involved in export, import or sequestration of the metal. CsoR is a copper(I) specific transcriptional regulator of the cso operon which consists of a putative copper export pump, CtpV. In copper limiting conditions, CsoR binds the operator/promoter region of the cso operon. In increased concentrations of copper (I), CsoR binds copper (I) with high affinity and is released from the operator/promoter site, causing derepression of the cso operon. To gain structural insight into CsoR function, we have solved the crystal structure of copper(I) bound CsoR. The structure reveals a homodimer with a subunit bridging copper site. The trigonal planar geometry and the presence of cysteine and histidine ligands at the metal site are favorable for copper(I) binding. The structure reveals a novel DNA binding fold in CsoR, making it the founding member of a new structural class of metalloregulators.

Thesis (Ph.D.)--Virginia Commonwealth University, 2007. / Title from title-page of electronic thesis. Prepared for: Dept. of Biochemistry. Bibliography: leaves 125-142

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第17780号 / 医博第3806号 / 新制||医||999(附属図書館) / 30587 / 京都大学大学院医学研究科医学専攻 / (主査)教授 妻木 範行, 教授 武藤 学, 教授 松田 道行 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The localization and translation of messenger ribonucleic acids (mRNAs) play crucial roles in cellular function and diseases, and are regulated by numerous RNA-binding proteins (RBPs) and small non-coding RNAs, called trans-acting factors. Biochemical and imaging methods used to study RNA interactions with these trans-acting elements have made important discoveries in characterizing how these factors regulate gene expression and determining the RNA sequence to which they bind. However, the spatiotemporal information regarding these interactions in subcellular compartments have been difficult to determine or to quantify accurately. To image and quantify native RNA and RNA–protein interactions simultaneously in situ, we developed a proximity ligation assay that combines peptide-modified RNA imaging probes. It can detect the RNAs in live cells and the interactions at a single-interaction level. Lastly, it can produce results that are easily quantifiable. We tested the specificity and sensitivity of this technique using two models: interactions between the genomic RNA and the N protein of human respiratory syncytial virus as well as those between exogenous transcripts with or without the Human antigen R (HuR) binding site and HuR. To validate this method, its accuracy and utility have been demonstrated in three models: poly(A)+ or β-actin mRNAs binding to different cytoskeleton for localization, poly(A)+ or β-actin mRNAs interacting with HuR for stabilization, and programmed cell death 4 (PDCD4) mRNA binding to HuR or T-cell intracellular antigen (TIA1) for translational regulation.