Organisms are constantly challenged by the surrounding environment and alter their physiology accordingly. Some environment-induced changes in one generation are inherited in the offspring, and this long-lasting memory of parental experience has gained a lot of attention recently due to its implications in the organism's development and health. One example is transmission of RNAi-induced silencing from parents to progeny in C. elegans. Although this phenomenon has been known for more than a decade, the parental contribution to RNAi inheritance is still unclear. Here, we show that the nuclear hormone receptor DAF-12 mediates a cross-generational signaling that regulates RNAi in zygotes. Pol II ChIP-qPCR revealed that normally, DAF-12 enhances transcriptional repression induced by RNAi. Mutant analysis demonstrated that the role of DAF-12 in RNAi is distinct from its function in developmental timing or heterochronic pathways. Surprisingly, DAF-12 acts in mothers to alter the RNAi efficiency in zygotes, indicating the presence of mother-to-offspring, DAF-12-dependent signals that enhance RNAi in zygotes. Considering the previous studies showing that the function of DAF-12 is determined by environmental cues, we tested and found that the role of DAF-12 in RNAi enhancement in zygotes depends on the environmental cues presented to mothers during their development. These results demonstrate a novel role of DAF-12 as a modulator of RNAi and its contribution to cross-generational signaling. Moreover, the findings imply a potential interaction between environmental conditions and small RNA pathways.

Sorghum (Sorghum bicolor L. Moench) ranks fifth worldwide in production among cereals. It is a major staple food for millions in Africa and Asia, and a major livestock feed grain in developed countries. However, the sorghum grain is poor in lysine content, limiting its value as food and feed. In this study, I hypothesize that reduction of some of the major storage proteins that are inherently poor in lysine through in vitro manipulation will result in the enhanced expression of proteins with a better lysine profile and, thus, increased overall grain lysine content. Sorghum genotypes were screened for in vitro amenability and a sorghum genotype-tissue culture medium combination that yielded the highest somatic embryo callus formation and regeneration potential, was identified. This resulted in the establishment of a sorghum biolistic transformation method with a transformation efficiency of 3.36%, the highest reported to date. Using genetic engineering tools, the enhancement of the nutritional quality of grain sorghum was achieved by increasing the seed lysine content. An RNAi co-suppression strategy was employed and resulted in 45.23 and 77.55% increase in whole seed and endosperm lysine increase, respectively. The co-suppression RNAi constructs targeted the endosperm specific suppression of three lysine-poor storage proteins, namely ä-kaf-2, ã-kaf-1 and -2, and an enzyme that catalyzes seed lysine degradation, lysine keto-gluterate reductase (LKR). Seven independent transgenic events displayed successful transgene integration for both the selectable marker gene and the target constructs. However, the Southern blot hybridization analysis revealed two transgenic events that displayed transgene re-arrangement at the 5’promoter end, thus resulting in a lack of suppression of target proteins. Variations in target proteins co-suppression was observed with Western blot analysis and RT-PCR for both the target kafirins and LKR suppression, and no lysine improvement was observed where no kafirin suppression occurred. The transgenic co-suppression of the target kafirins resulted in the endosperm structural change from a hard, corneous endosperm to a soft, floury endosperm, consistent with ã-zein suppression in the Opaque-2 maize mutant. / Thesis (PhD)--University of Pretoria, 2010. / Plant Science / unrestricted

Amyotrophic lateral sclerosis (ALS) is a devastating adult onset neurodegenerative disorder characterized by the selective degeneration of upper and lower motor neurons. Patients typically die from respiratory failures within 2-5 years after diagnosis. One of the milestones in ALS research is the discovery Fused in Sarcoma (FUS), an ALS causative gene. FUS is an RNA/DNA-binding protein and predominantly resides in the nucleus. Majority of the FUS mutations are located in the C-terminus and causing aberrant misdistribution to the cytoplasm. Currently, only a few binding partners of FUS are known, which makes it difficult to speculate on the function and interaction of the protein. In this study, we conducted a kinome-wide RNAi screen to identify kinases that affect the localization of FUS. A dual specificity protein kinase named CDC2-like kinase (CLK1) from the screen was found to be responsible for in post-translational modification of FUS and affects the localization of FUS in the nucleus. The identification of CLK1 as FUSmodifying kinase is consistent with roles ascribed to both in the binding and regulation of RNA.

Myotonic Dystrophy type 1 (DM1), the most common form of adult muscular dystrophy (~1:8000) currently has no effective treatment. In DM1, expansion of a tri-nucleotide repeat in the 3' UTR of the DMPK gene results in DMPK mRNA hairpin structures, aggregating as insoluble ribonuclear foci. The resulting mis-regulation of important splicing factors, causes the inclusion of fetal exons in dozens of transcripts that contribute to the disease phenotype. In order to identify novel gene targets and kinase signalling pathways for potential therapeutics we have performed a high-throughput RNAi. RNA foci were visualized and quantified by in-situ hybridization. From our screen, we have identified a novel gene, PACT, as a modulator of foci integrity and that PACT knockdown can induce MBNL1 protein levels. The identified signalling complex represents a valid target for DM1 therapeutics. Our data further emphasizes the utility of RNAi screens in identifying disease-associated genes.

Assessing the risk of transgenic crop products is essential when determining the safety of a crop for deregulation and commercialization. The Organization of Economic and Cooperative Development (OECD), International Standards Organization (ISO), and governmental regulatory agencies require a battery of tests to demonstrate the safety of a GM product against several surrogate species of organisms that perform various ecosystem services. Assays are performed using toxicology methods established for pesticides. These methods have been applied to testing the safety, specificity and fate of Bacillus thuringiensis (Bt) Cry protein toxins engineered into crop plants and information exists on the effects on non-target organisms (NTOs). Toxicology assays are typically evaluated through a tier-based approach, where, if no or negligible risk via oral toxicity or phenotypic changes then a risk decision can be made. Long term exposure studies are often performed after commercial release of the crop occurs and provide a more in depth understanding of environmental impacts. Risk analyses are currently being performed on the product of the next generation of GM crops that express dsRNAs against Western Corn Rootworm. This thesis provides another such study, primarily focused on Folsomia candida, a microarthropod that is the subject of numerous toxicological studies. I describe the development of dsRNA stability assays, which indicate stability of dsRNA across assay time, both with and without F. candida. When exposed to dsRNA levels several orders of magnitude higher that what would be encountered in the environment, F. candida is not negatively impacted as seen through gene expression and life history trait analysis.

Cystic fibrosis transmembrane conductance regulator (CFTR) is an anion channel that when mutated causes the disease cystic fibrosis (CF). Many obstacles hinder the understanding of CF disease pathogenesis, impeding advancements in understanding how mutations cause disease, and slowing the progress towards new treatments. To this end, we have profiled the transcriptome (mRNA and microRNA) of human and newborn pig CF and non-CF airway epithelia. We show that the use of cross-species transcriptomics allows the identification of genes differentially expressed owing to the loss of CFTR, and not due to confounding environmental or secondary disease progression influences. The identification of reduced OAS1 expression in CF samples is a case in point. We also demonstrate the utility of transcriptome profiling and longitudinal studies in pigs, providing greater understanding of the molecular mechanisms underlying CF disease progression. MicroRNAs (miRNAs) comprise a large family of ~21-nt long non-coding RNAs that function as key post-transcriptional regulators of gene expression. Very little is known of how CFTR is regulated in the cell, both transcriptionally and post-transcriptionally. We discovered three miRNAs: miR-509-3p, miR-494 and miR-138 with possible CFTR regulatory functions. miR-509-3p or -494 directly target the CFTR mRNA, and decrease CFTR levels when over expressed; while inhibiting them had the opposite effect. Upon stimulating human airway epithelial cells with TNFα or IL-1β, we observed an increase in expression of both miRNAs mediated in part by the NF-κB transcription factor complex, with a concurrent decrease in CFTR expression. Gene ontology classification of predicted targets of miR-509-3p and/or miR-494 expressed in the airway epithelium revealed enrichment for genes in ion transport pathways. To our knowledge, this is the first suggestion of a possible role for miRNAs regulating a broad range of important epithelial electrolyte and fluid transport proteins. The study of miR-138 mediated regulation of CFTR expression has led to novel discoveries in the field of CFTR transcriptional control. We discovered SIN3A to be a novel transcriptional repressor of CFTR, interacting with CTCF on the CFTR promoter at the -20.9 kb DHS. By validating SIN3A as a conserved target of miR-138, we also discovered miR-138 to be a novel transcriptional regulator/activator of CFTR. The most common CFTR mutation, ΔF508, causes protein misfolding, degradation, and CF. Manipulating the miR-138/SIN3A regulatory network improved the biosynthesis of CFTR-ΔF508, restoring Cl- transport to human CF airway epithelia. To our knowledge, this is the first example of an individual miRNA having such broad regulatory functions. This discovery also provided novel targets for restoring CFTR function in cells affected by the most common CF mutation. To this end, we are utilizing the molecular signatures of miR-138 over-expression and SIN3A knockdown to identify candidate genes for RNA interference screens, and to identify candidate small molecule drugs that might mimic the effects of these two interventions. The goal of this approach is to develop a new therapeutic agent that restores anion transport to airway epithelia and other cell types and tissues affected by CF.

RNA interference (RNAi) is a pathway, which employs Dicer to process long double stranded RNAs (dsRNA) from endogenous or exogenous sources into short interfering RNAs (siRNA). siRNAs are loaded onto Argonaute proteins to mediate sequence-specific post-transcriptional RNA targeting resulting in regulation of protein-coding genes and retrotransposons or antiviral immune response. Another small RNA pathway - PIWI-associated RNA (piRNA) pathway is suppressing retrotransposons in the germline. In mice, canonical RNAi pathway activity is negligible in somatic cells where a full-length Dicer produces gene-regulatory microRNAs (miRNA) but RNAi is highly active in oocytes, which express a truncated oocyte-specific Dicer isoform (DicerO ). DicerO lacks an N-terminal DExD helicase domain and has higher cleavage activity of long dsRNAs. Deletion of oocyte specific DicerO promoter leads to transcriptome aberrations, which include upregulation of putative RNAi targets and MT retrotransposons and, consequently, to meiotic spindle defects and female sterility. In contrast, the piRNA pathway is non-essential in mouse oocytes, potentially because of overlapping functions of RNAi. The PhD thesis aims to understand biological significance of mammalian endogenous RNAi and to explore consequences of re-activated RNAi...

RNA interference (RNAi) is widely used to specifically silence the expression of any gene to study its function and to identify and validate therapeutic targets. Despite the popularity of this technology, recent studies have shown that RNAi may also silence non-targeted genes. Here we demonstrate the utility of a quick, efficient and robust approach to directly validate the specificity of RNAi as an alternative to indirect validation of RNAi through gene expression profiling. Our approach involves reversing (complementing) the RNAi-induced phenotype by introducing a synthetic version of the target gene that is designed to escape silencing. This synthetic gene complementation approach can also be used for mutational analysis of the target gene, or to provide a functional version of a defective protein after silencing the defective gene by RNAi. Using this approach we demonstrate that the loss of systemic acquired resistance, a form of innate immunity in plants, is indeed due to the silencing of salicylic acid-binding protein 2 rather than to off-target effects.

ZUSAMMENFASSUNG Obwohl diverse Mutationen des NF-κB Systems in Myelomzelllinien und primären Myelomzellen eine pathogenetische Beteiligung andeuten, ist die Relevanz des IKK Komplexes als molekularer Angriffspunkt für die Entwicklung medikamentöser Therapieoptionen noch nicht ausreichend geklärt. Zwar führte die Applikation des IKK-β Inhibitors MLN120b dosisabhängig und längerfristig zu einer Reduktion der Zellviabilität in einer Vielzahl von Myelomzelllinien, doch kann nicht ausgeschlossen werden, dass bei höheren Konzentrationen unspezifische Wirkungen für die beobachteten Effekte (mit-) verantwortlich sind. Aus diesem Grund erfolgte in der vorliegenden Arbeit eine spezifische Suppression von IKK-α, IKK-β oder IKK-γ mittels transienter Transfektion von shRNA Expressionsvektoren oder Stealth-siRNA. Es folgte die Charakterisierung der verminderten Zielproteinspiegel mittels Western-Blot und die Messung der Viabilität der Zellen mittels FACS Analysen. Darüber hinaus wurde in TNF-α Stimulationsexperimenten der Effekt der Suppression von IKK-β mittels Stealth-siRNA auf (Phospho-)IκB-α analysiert. Schließlich erfolgte die Applikation des IKK-β Inhibitors TPCA, dessen Wirkung auf die Zellviabilität und auf die TNF-α-vermittelte Phosphorylierung und Degradation von IκB-α in MM.1S Zellen untersucht wurde. Die Experimente mit Stealth-siRNA zeigten, dass weder die Suppression von IKK-β, noch die Suppression von IKK-α oder IKK-γ in AMO-1, L363 oder MM.1S eine Verminderung der Zellviabilität bewirken konnte. Auch eine kombinierte Suppression von IKK-α zusammen mit IKK-β in L363 und MM.1S Zellen bewirkte keinen vermehrten Zelltod. Dagegen zeigte die Behandlung von MM.1S Zellen mit hohen Konzentrationen von TPCA einen geringen Effekt auf das Überleben dieser Zellen. Die Suppression von IKK-β mittels Stealth-siRNA in MM.1S konnte nicht die TNF-α vermittelte IκB-α Phosphorylierung und Degradation verhindern. Sowohl die hohe TNF-α Konzentration von 100ng/ml, als auch eine unvollständige Suppression von IKK-β könnte dazu beigetragen haben. In analogen Experimenten mit TPCA konnte die TNF-α vermittelte IκB-α Phosphorylierung und Degradation dagegen effektiv unterdrückt werden. In der Zusammenschau der Ergebnisse konnte somit eine potenzielle therapeutische Relevanz des IKK-Komplexes als molekularer Angriffspunkt für eine Myelomtherapie nicht gefunden werden. Eine noch detailliertere Analyse der Funktionalität des Signalwegs (insbesondere eine Messung der Aktivität der NF-κB Transkriptionsfaktoren im Zellkern) und die Etablierung stabiler und induzierbarer Expressionssysteme für längerfristige Untersuchungen der RNAi Wirkungen in Myelomzellen, stellen weiterführende Wege zu einer umfangreicheren Beurteilung der pathobiologischen und therapeutischen Bedeutung des NF-κB Systems dar. Darüber hinaus sind die das NF-κB System betreffenden Mutationen genauer hinsichtlich ihrer potenziellen Wirkung auf NF-κB unabhängige Signalwege zu untersuchen. / ABSTRACT Although the known mutations in NF-kB in imply to view the IKK complex to be involved in promoting survival of Multiple Myeloma cells, and IKK-beta inhibitors have been reported to show anti-Myeloma activity, the actual significance of IKK as a molecular target for treatment of the desease remains unclear. This is due to the fact that the influence on survival by pharmacological inhibitors on many Multiple Myeloma cells has been shown to be dependend on high concentration applications and on long-term incubation times, both inheriting the possibility of unspecific drug effects. Therefor we chose an alternative approach and performed a RNAi mediated knockdown of each of the three components of the IKK Complex (IKK-alpha, IKK-beta and IKK-gamma) and measured the survival rate of the Multiple Myeloma celllines AMO-1, L363 and MM.1s. None of the knockdowns led to a decrease of Multiple Myeloma survival rate, and even a combined knockdown of IKK-alpha and IKK-beta in L363 and MM.1S showed significant effects on the survival. Summarized, IKK is not crucially involved in promoting survival of the mentioned Multiple Myeloma celllines and thus the relevance of the complex as a therapeutic target for the treatment of Multiple Myeloma is not thoroughly clarified yet. Further investiations in different celllines and RNAi mediated knockdowns in longer time intervalls might resolve the true significance of IKK for Myeloma survival.

京都大学 / 0048 / 新制・論文博士 / 博士(医科学) / 乙第13169号 / 論医科博第4号 / 新制||医科||6(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 近藤 玄, 教授 高田 穣 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM