<p>In Drosophila melanogaster, where males are XY and females are XX, dosage compensation is acheived by approximately two-fold upregulation of transcription of the single male X chromosome. This upregulation is mediated by the dosage compensation complex (DCC), which is assembled in a sequential manner on the male X chromosome and is composed of the two noncoding roX (RNA on the X) RNAs and at least five proteins, including the RNA helicase Maleless (MLE). MLE contains two highly conserved double stranded RNA binding domains (DRBDs) at the N terminus. We investigated the roles of the roX RNAs and MLE helicase through experiments using classical genetic methods to generate and analyze the effects of mutants and mutant transgenes, immunolocalization experiments to study MSL protein and roX RNA to chromosomes. For the first time in vivo, we demonstrate that MLE associates with double stranded RNA in a sequence non-specific manner that is independent of other DCC components. Importantly, we find that the DSRBDs of MLE are essential for dosage compensation but are not required for MLE localization to the male X chromosome. We propose that although the DSRBDs are not essential for ds RNA binding, they may act synergistically with other domains of MLE or other MSLs to associate with RNA in vivo. We propose that a MLE/ roX RNA association involving secondary structure formed by the roX RNAs may be involved in the assembly, stabilization, or function of the DCC.</p> / Dissertation

Viral double-stranded RNA is a key pathogen invasion signal recognized by the human innate immune system. All adenoviruses synthesize at least one highly structured RNA (VAI) to suppress this antiviral response by attenuating the activity of antiviral proteins. Surprisingly, VAI RNA was previously shown to positively regulate the activity of one interferon-inducible antiviral protein, 2’-5’-oligoadenylate synthetases (OAS). The present thesis focuses on investigating the regulation of a human OAS1 isoform by VAI RNA and its derivatives. An Escherichia coli protein expression and purification system has been developed for OAS1 protein production. A combination of biochemical and biophysical approaches was employed to examine VAI RNA binding affinity, activation potential for OAS1 and OAS1:VAI RNA complex formation. Taken together, I have found that while full-length VAI does indeed activate OAS1 in vitro, a truncated version lacking the terminal stem has the opposite effect, and this is the physiologically important response.

Viral double-stranded RNA is a key pathogen invasion signal recognized by the human innate immune system. All adenoviruses synthesize at least one highly structured RNA (VAI) to suppress this antiviral response by attenuating the activity of antiviral proteins. Surprisingly, VAI RNA was previously shown to positively regulate the activity of one interferon-inducible antiviral protein, 2’-5’-oligoadenylate synthetases (OAS). The present thesis focuses on investigating the regulation of a human OAS1 isoform by VAI RNA and its derivatives. An Escherichia coli protein expression and purification system has been developed for OAS1 protein production. A combination of biochemical and biophysical approaches was employed to examine VAI RNA binding affinity, activation potential for OAS1 and OAS1:VAI RNA complex formation. Taken together, I have found that while full-length VAI does indeed activate OAS1 in vitro, a truncated version lacking the terminal stem has the opposite effect, and this is the physiologically important response.

Double-stranded RNA (dsRNA), a double helix formed by two antiparallel complementary RNA strands, is a unique structure with a variety of biological effects. dsRNA can be introduced into the cell from exogenous sources or it can be produced endogenously. There are four basic mechanisms producing dsRNA: inverted repeat transcription, convergent transcription, pairing of sense and antisense RNAs produced in trans, and RNA dependent RNA polymerase-mediated synthesis dsRNA. Different mechanisms of production determine additional structural features of dsRNA, such as dsRNA termini, mismatches etc. These features may affect cellular response to dsRNA. Recognition of dsRNA can trigger several responses that act in sequence-specific or sequence-independent manners. The main sequence- specific response triggered by dsRNA is RNA interference (RNAi) is. Our laboratory has been studying mechanism of induction of RNAi in mammalian cells using one specific type of long dsRNA expression system. The dsRNA used in these experiments formed hairpin structure with long 5' and 3' single-strand RNA overhangs. We hypothesized that other dsRNA substrates might be more efficient than the one used in mammalian RNAi experiments since 2002. Accordingly, the main aim of my thesis was to compare efficiency of different dsRNA...

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第22135号 / 生博第422号 / 新制||生||55(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 藤田 尚志, 教授 朝長 啓造, 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

DNA double strand breaks (DSB) are cytotoxic lesions that can lead to genome rearrangements and genomic instability, which are hallmarks of cancer. The two main DSB repair pathways are non-homologous end joining and homologous recombination (HR). While HR is generally highly accurate, it has the potential for gross chromosomal rearrangements (GCRs) that occur directly or through intermediates generated during the repair process. Whole genome sequencing of cancers has revealed numerous types of structural rearrangement signatures that are often indicative of repair mediated by sequence homology. However, it can be challenging to delineate repair mechanisms from sequence analysis of rearrangement end products from cancer genomes, or even model systems, because the same rearrangements can be generated by different pathways. Numerous studies have provided insights into the types of spontaneous GCRs that can occur in various Saccharomyces cerevisiae mutants. However, understanding the mechanism and frequency of formation of these GCR without knowledge of the initiating lesions is limited. Here, we focus on DSB-induced repair pathways that lead to GCRs. Inverted duplications occur at a surprisingly high frequency when a DSB is formed near short inverted repeats in cells deficient for the nuclease activity of Mre11. Similar to previously proposed models, the inverted duplications occur through intra-strand foldback annealing at resected inverted repeats to form a hairpin-capped chromosome that is a precursor to dicentric chromosomes. Surprisingly, we found that DNA polymerase δ proof-reading activity but not the Rad1-Rad10 nuclease is required for inverted duplication formation, suggesting a role for Pol δ in the removal of the heterologous tails formed during foldback annealing. Contrary to previous work on spontaneous inverted duplications, we find that DSB-induced inverted duplications require the Pol δ processivity subunit Pol32 and that RPA plays little role in their inhibition, suggesting that spontaneous inverted duplications arise differently than those induced by DSBs. We show that stabilization of dicentric chromosomes after breakage involves telomere capture through a strand-invasion step mediated by repeat sequences and requires Rad51. Previous work on spontaneous inverted duplications suggested that Tel1, but not Mre11-Sae2, inhibits inverted duplications that initiate from inverted repeats separated by long spacers (> 12 bp). However, we do not find evidence for this requirement. Cells with Tel1 deletion can still resolve hairpins containing loops up to 30 nt long. Furthermore, deletion of Sae2, but not Tel1, increases the frequency of inverted duplications when a DSB is induced near an inverted repeat separated by a 20 bp-long spacer. This highlights another difference between spontaneous and DSB-induced GCRs. Finally, we find that the sequence context of a DSB affects the type of GCR outcome. Inverted repeats are required for the formation of inverted duplications, as the deletion of a DSB-proximal inverted repeat significantly reduces the incidence of this type of rearrangement. Furthermore, introduction of a DSB near short telomere-like sequence is required for chromosome truncations stabilized by de novo telomere addition. The effect of the sequence context can partly explain how repair pathways can be channeled to different mutagenic outcomes. Our results highlight the importance of considering how the initiating lesion can affect the type of resulting GCRs and the mechanisms by which they occur.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20782号 / 生博第388号 / 新制||生||51(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 藤田 尚志, 教授 朝長 啓造, 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

A strain of Trichomonas vaginalis infected with a double-stranded RNA virus showed pronounced cytopathology in the form of giant syncytia generated by the recruitment of single cells. The giant cells ultimately lysed, releasing virus into the culture medium. In the infected cells, clusters of electron-dense particles resembling viral structures were found in the cytoplasm. In addition, distinctive inclusions composed of similar particles were present in the nuclei of some cells. Double-stranded viral RNA of 5.5 kbp was demonstrated in both cytoplasmic and nuclear fractions from these cells. Viral particles collected from the cell-free culture supernatant were of the same shape and size as the RNA virus isolated from a strain of T. vaginalis described previously (Wang and Wang, Journal of Biological Chemistry, 260: 3697-3702, 1985; Wang and Wang, Proceedings of the National Academy of Sciences of the U.S.A. 83: 7956-7986) which does not show this cytopathology.

No description available.

Double-stranded RNA (dsRNA) is a key molecule that initiates the immune response to viral infection, but increasingly endogenous (self) dsRNA has been found to be central to the pathology of diverse non-infectious diseases, from neurodegenerative disease to autoimmunity to cancers. Therefore, it is critical to understand the mechanisms that regulate endogenous dsRNA and the pattern recognition receptors that sense dsRNA. In this dissertation, I address three main questions pertaining to this. First, why is the brain so prone to dsRNA-mediated non-infectious disease, especially considering that dsRNA sensors are expressed in almost all tissues. Using stem cell differentiation, gene expression manipulation, and microscopy, I determined that neurons are a special cell type that express high levels of endogenous dsRNA. This high dsRNA burden in neurons is driven by global lengthening of 3`untranslated regions (3`UTRs) and induces tonic inflammation. Second, I examined the mechanism through which the dsRNA regulator ADAR1 controls endogenous dsRNA levels. I employed heavy use of tissue culture and visualization of dsRNA by confocal microscopy to determine that both the dsRNA-binding and dsRNA-editing activities of ADAR1 are required to suppress global endogenous dsRNA levels. Third, after identifying the existence of transcript isoforms of the key dsRNA sensor PKR, I explored their regulatory potential on the PKR protein itself. By genetically altering human cells, I identify that 3`UTR isoforms of PKR regulate transcript localization, translation efficiency, and PKR protein activatability. Overall, the studies described herein demonstrate novel regulatory roles of endogenous dsRNA and underscore the importance of dsRNA in neurological disease.