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Die Weinkanton Luzern boomt. Die Region lebt von einer hohen Innovationskraft, spannenden Weinen und einem wachsenden Markt. Mehr als jeder dritte Rebstock ist eine Piwi-Sorte. Im Herzen der Schweiz wird Weinbau zukunfsorientiert und auf Basis neuester Wärmesummen-Berechnungen der Zürcher Hochschule für Angewandte Wissenschafen (ZHAW) betrieben.

Background: PIWI-interacting RNAs (piRNAs) are an abundant single-stranded type of small non-coding RNAs (sncRNAs), which initially were discovered in gonadal cells, with a role as defenders of genomic integrity in the germline, acting against the transposable elements. With a regular size range of 21-35 nt, piRNAs are associated with a PIWI-clade of Argonaute family proteins. The most widely accepted mechanisms of biogenesis for piRNAs involve the transcription of longer precursors of RNAs to be processed, by complexes of proteins, to functional size, preferentially accommodating uridine residues at the 5’ end and 3’ methylation to increase the stability of these molecules. piRNAs have also been detected in somatic cells, with diverse potential functions, indicating their high plasticity and pleiotropic activity. Discovered about two decades ago, piRNAs are a large and versatile type of sncRNA and that remain insufficiently identified and analyzed, through next-generation sequencing (NGS), to evaluate their processing, functions, and biogenesis in different cell types and during development. piRNAs’ distinction from other sncRNAs has led to controversial results and interpretation difficulties when using existing databases because of the heterogeneity of the criteria used in making the distinction., Description: We present “piRNA-IPdb”, a database based uniquely on datasets obtaining after the defining characteristic of piRNAs: those small RNAs bound to PIWI proteins. We selected and analyzed sequences from piRBase that exclusively cover the binding to PIWI. We pooled a total of 18,821,815 sequences from RNA-seq after immunoprecipitation that included the binding to any of the mouse PIWI proteins (MILI, MIWI, or MIWI2)., Conclusions: In summary, we present the characteristics and potential use of piRNA-IPdb database for the analysis of bona fide piRNAs.

P-element induced wimpy testis (PIWIs) are crucial guardians of genome integrity, particularly in germ cells. While mammalian PIWIs have been primarily studied in mouse and rat, a homologue for the human PIWIL3 gene is absent in the Muridae family, and hence the unique function of PIWIL3 in germ cells cannot be effectively modeled by mouse knockouts. Herein, we investigated the expression, distribution, and interaction of PIWIL3 in bovine oocytes. We localized PIWIL3 to mitochondria, and demonstrated that PIWIL3 expression is stringently controlled both spatially and temporally before and after fertilization. Moreover, we identified PIWIL3 in a mitochondrial-recruited three-membered complex with Tudor and KH domain-containing protein (TDRKH) and poly(A)-specific ribonuclease-like domain containing 1 (PNLDC1), and demonstrated by mutagenesis that PIWIL3 N-terminal arginines are required for complex assembly. Finally, we sequenced the piRNAs bound to PIWIL3-TDRKH-PNLDC1 and report here that about 50% of these piRNAs map to transposable elements, recapitulating the important role of PIWIL3 in maintaining genome integrity in mammalian oocytes.

Transposable elements (TEs) can damage genomes, thus organisms use a variety of mechanisms to repress TE expression. The PIWI-piRNA pathway is a small RNA pathway that represses TE expression in the germline of animals. Here we explore the function of the pathway in the somatic stem cells of Hydra, a long-lived freshwater cnidarian. Hydra have three stem cell populations, all of which express PIWI proteins; endodermal and ectodermal epithelial stem cells (ESCs) are somatic, whereas the interstitial stem cells have germline competence. To study somatic function of the pathway, we isolated piRNAs from Hydra that lack the interstitial lineage and found that these somatic piRNAs map predominantly to TE transcripts and display the conserved sequence signatures typical of germline piRNAs. Three lines of evidence suggest that the PIWI-piRNA pathway represses TEs in Hydra ESCs. First, epithelial knockdown of the Hydra piwi gene hywi resulted in up-regulation of TE expression. Second, degradome sequencing revealed evidence of PIWI-mediated cleavage of TE RNAs in epithelial cells using the ping-pong mechanism. Finally, we demonstrated a direct association between Hywi protein and TE transcripts in epithelial cells using RNA immunoprecipitation. Altogether, our data reveal that the PIWI-piRNA pathway represses TE expression in the somatic cell lineages of Hydra, which we propose contributes to the extreme longevity of the organism. Furthermore, our results, in combination with others, suggest that somatic TE repression is an ancestral function of the PIWI-piRNA pathway.

Germcells are responsible for the transmission of all genetic information from one generation to the next and engage a genome defense pathway that is based upon RNA interference. This socalled PIWI or piRNA pathway protects the genome by targeting transposon transcripts and renders them inactive. PIWI proteins and their interacting small RNAs, the piRNAs, therefore represent an ancient defense system against intergenomic parasites. In zebrafish, where germcells are specified by the inheritance of germplasm, information about maternal transposon activity is passed on via the maternally provided PIWI protein Ziwi, a component of germ plasm. This maternally provided piRNA pool is thought to represent a first line of defense against transposon activity in early germ cells. Little is known about what happens in early germ cells of zebrafish in the time between their arrival at the genital ridge and the time when a functional gonad is established. We find that in this timeframe primordial germ cells (PGCs) are going through a transition from a maternal to a zygotic program. This change is accompanied by massive morphological changes and the induction of a germline specific transcriptional program. This not only leads to the expression of germline genes but also large intergenetic regions, start to get expressed. PiRNAs produced in the adult gonads map to these regions, suggesting that they might represent piRNA clusters, similar to what is described in mouse and drosophila. In the first few days, not only these clusters become transcribed but also the piRNA pathway becomes active leading to the amplification of maternal piRNAs. The second chapter of this thesis is looking at this early germ cell development. What happens in gonads where the PIWI pathway is compromised is subject to chapters 3 and 4. Vasa and Tdrd9 are two conserved proteins involved in the PIWI pathway in other organisms and we find that in zebrafish, absence of either of these two proteins leads to the l

RNA interference is a crucial antiviral mechanism in arthropods, including in mosquito vectors of arthropod-borne viruses (arboviruses). Although the exogenous small interfering RNA (siRNA) pathway constitutes an efficient antiviral response in mosquitoes, virus-derived P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) have been implicated in the response to alpha-, bunya- and flaviviruses in Aedes spp. mosquitoes. Culex mosquitoes transmit several medically important viruses including West Nile virus (WNV), but are considerably less well studied than Aedes mosquitoes and little is known about antiviral RNA interference in Culex mosquitoes. Therefore, we sequenced small RNA (sRNA) libraries from different Culex cell lines and tissues infected with WNV. The clear majority of virus-derived sRNA reads were 21 nt siRNAs in all cell lines and tissues tested, with no evidence for a role of WNV-derived piRNAs. Additionally, we aligned sRNA reads from Culex quinquefasciatus Hsu cells to the insect-specific rhabdovirus, Merida virus, which persistently replicates in these cells. We found that a significant proportion of the sRNA response to Merida virus consisted of piRNAs. Since viral DNA forms have been implicated in siRNA and piRNA responses of Aedes spp. mosquitoes, we also tested for viral DNA forms in WNV infected Culex cells. We detected viral DNA in Culex tarsalis cells infected with WNV and, to a lesser amount, WNV and Merida virus-derived DNA in Culex quinquefasciatus Hsu cells. In conclusion, Hsu cells generated Merida virus-derived piRNAs, but our data suggests that the major sRNA response of Culex cells and mosquitoes to WNV infection is the exogenous siRNA response. It is also evident that sRNA responses differ significantly between specific virus-mosquito combinations. Future work using additional Culex-borne viruses may further elucidate how virus-derived piRNAs are generated in Culex cells and what role they may play in controlling replication

Germcells are responsible for the transmission of all genetic information from one generation to the next and engage a genome defense pathway that is based upon RNA interference. This socalled PIWI or piRNA pathway protects the genome by targeting transposon transcripts and renders them inactive. PIWI proteins and their interacting small RNAs, the piRNAs, therefore represent an ancient defense system against intergenomic parasites. In zebrafish, where germcells are specified by the inheritance of germplasm, information about maternal transposon activity is passed on via the maternally provided PIWI protein Ziwi, a component of germ plasm. This maternally provided piRNA pool is thought to represent a first line of defense against transposon activity in early germ cells. Little is known about what happens in early germ cells of zebrafish in the time between their arrival at the genital ridge and the time when a functional gonad is established. We find that in this timeframe primordial germ cells (PGCs) are going through a transition from a maternal to a zygotic program. This change is accompanied by massive morphological changes and the induction of a germline specific transcriptional program. This not only leads to the expression of germline genes but also large intergenetic regions, start to get expressed. PiRNAs produced in the adult gonads map to these regions, suggesting that they might represent piRNA clusters, similar to what is described in mouse and drosophila. In the first few days, not only these clusters become transcribed but also the piRNA pathway becomes active leading to the amplification of maternal piRNAs. The second chapter of this thesis is looking at this early germ cell development. What happens in gonads where the PIWI pathway is compromised is subject to chapters 3 and 4. Vasa and Tdrd9 are two conserved proteins involved in the PIWI pathway in other organisms and we find that in zebrafish, absence of either of these two proteins leads to the l

PIWI Proteine sind die zentralen Darsteller eines RNA-basierten Mechanismus, der die Mobilisierung transponierbarer Elemente im Genom unterdrückt, um genetische Stabilität zu gewährleisten. Demzufolge sind PIWI-Proteine für die langfristige Erhaltung verschiedener Stamzellpopulationen notwendig. Beispiele dafür sind verschiedene adulte somatische Stammzellen in Drosophila und die Stammzellen der Keimbahn aller bisher untersuchten Tierarten. Bei Säugetieren sind die beschriebenen Funktionen von PIWI Proteinen strikt auf die männliche Keimbahn beschränkt. Trotz Andeutungen auf eine Rolle von PIWI-Proteinen in somatischen Zellen von Säugetieren, wurde eine Funktion bisher nicht beschrieben. Ähnlich wie Stammzellen, können sich Makrophagen in verschiedenen Geweben selbst-erneuern, um ihre Populationen zu erhalten. Diese Selbsterneuerung beruht auf der geringen Expression der Transkriptionsfaktoren MafB und cMaf, was die Aktivierung eines stammzell-ähnliches Gen-Netzwerk, das die Proliferation vorantreibt. Makrophagen mit einer genetischen Deletion von MafB und cMaf (MafDKO-Makrophagen) oder Makrophagen mit natürlich niedriger Expression von MafB oder cMaf, wie z.B. alveoläre Makrophagen, weisen dementsprechend eine erweiterte Kapazität zur Selbsterneuerung auf. Wie haben festgestellt, dass eine kurze Isoform des Maus- Gens Piwil2, die wir ‚Piwito’ genannt haben, in MafDKO und alveolären Makrophagen exprimiert wird. Die Expression von Piwito ist für die normale Selbsterneuerung der untersuchten Makrophagen notwendig, wie die in vitro und in vivo Untersuchungen darlegen. Eine Abwesenheit von Piwito in alveolären Makrophagen führt zu einer Verkürzung derer Lebenspanne in Kultur. Außerdem beweisen wir, dass Piwito von MafB in nicht-proliferierenden Makrophagen gebunden und unterdrückt wird. Diese Studie ist somit der erste Bericht über eine somatische Funktion von PIWI-Proteinen in nicht transformierten Zellen von Säugetieren., PIWI proteins are the main players of an RNA-based gene regulatory machinery that represses transposable elements in the genome to prevent their mobilization and ensure genetic stability. PIWI proteins have thus highly conserved stem-cell functions. They are indispensable for the long-term maintenance of the somatic stem cells that drive regeneration in invertebrates, of various adult somatic stem cells in Drosophila and, most prominently, of the germline of all species studied so far. In mammals, their described functions are strictly restricted to the male germline. Despite suggestive observations for a role of PIWI proteins in the mammalian soma, robust evidence remains absent. Similar to stem cells, tissue macrophages can locally self-renew to maintain their populations. Mechanistically, their self-renewal relies on low expression of the macrophage transcription factors MafB and cMaf, since it allows the induction of a stem cell-like network of genes that drives proliferation. Macrophages with a genetic deletion of MafB and cMaf (MafDKO macrophages) acquire therefore the capacity to self-renew, defined by an indefinite growth in culture that does not comprise their identity and does not involve cancerogenic transformation. Similarly, macrophages with naturally low levels of MafB or cMaf, such as alveolar macrophages, display an extended self-renewal capacity in vivo and in vitro. We have found that a short isoform of the murine Piwil2 gene, that we named ‘Piwito’, is expressed in MafDKO and alveolar macrophages. Piwito expression is necessary for the unaltered self-renewal of macrophages, as shown by in vitro and in vivo assays. To highlight is the fact that Piwito deficiency limits the extended lifespan of alveolar macrophages in culture. Additionally, we show that Piwito is bound and repressed by MafB in quiescent macrophages. This study thus represents the first report of a somatic function for mammalian PIWI proteins in non-transformed cells.

Nach dem rekordwarmen Jahr 2018 war es nur logisch, dass der Vormittag des Rebbautags den Klimawandel zum Thema hatte. Am Nachmittag wurden die wichtigsten Label für nachhaltige Produktion präsentiert. Der Weinbereitungstag war der Vinifikation und Vermarktung der "neuen Rebsorten" gewidmet.

Germcells are responsible for the transmission of all genetic information from one generation to the next and engage a genome defense pathway that is based upon RNA interference. This socalled PIWI or piRNA pathway protects the genome by targeting transposon transcripts and renders them inactive. PIWI proteins and their interacting small RNAs, the piRNAs, therefore represent an ancient defense system against intergenomic parasites. In zebrafish, where germcells are specified by the inheritance of germplasm, information about maternal transposon activity is passed on via the maternally provided PIWI protein Ziwi, a component of germ plasm. This maternally provided piRNA pool is thought to represent a first line of defense against transposon activity in early germ cells. Little is known about what happens in early germ cells of zebrafish in the time between their arrival at the genital ridge and the time when a functional gonad is established. We find that in this timeframe primordial germ cells (PGCs) are going through a transition from a maternal to a zygotic program. This change is accompanied by massive morphological changes and the induction of a germline specific transcriptional program. This not only leads to the expression of germline genes but also large intergenetic regions, start to get expressed. PiRNAs produced in the adult gonads map to these regions, suggesting that they might represent piRNA clusters, similar to what is described in mouse and drosophila. In the first few days, not only these clusters become transcribed but also the piRNA pathway becomes active leading to the amplification of maternal piRNAs. The second chapter of this thesis is looking at this early germ cell development. What happens in gonads where the PIWI pathway is compromised is subject to chapters 3 and 4. Vasa and Tdrd9 are two conserved proteins involved in the PIWI pathway in other organisms and we find that in zebrafish, absence of either of these two proteins leads to the l

PIWI Proteine sind die zentralen Darsteller eines RNA-basierten Mechanismus, der die Mobilisierung transponierbarer Elemente im Genom unterdrückt, um genetische Stabilität zu gewährleisten. Demzufolge sind PIWI-Proteine für die langfristige Erhaltung verschiedener Stamzellpopulationen notwendig. Beispiele dafür sind verschiedene adulte somatische Stammzellen in Drosophila und die Stammzellen der Keimbahn aller bisher untersuchten Tierarten. Bei Säugetieren sind die beschriebenen Funktionen von PIWI Proteinen strikt auf die männliche Keimbahn beschränkt. Trotz Andeutungen auf eine Rolle von PIWI-Proteinen in somatischen Zellen von Säugetieren, wurde eine Funktion bisher nicht beschrieben. Ähnlich wie Stammzellen, können sich Makrophagen in verschiedenen Geweben selbst-erneuern, um ihre Populationen zu erhalten. Diese Selbsterneuerung beruht auf der geringen Expression der Transkriptionsfaktoren MafB und cMaf, was die Aktivierung eines stammzell-ähnliches Gen-Netzwerk, das die Proliferation vorantreibt. Makrophagen mit einer genetischen Deletion von MafB und cMaf (MafDKO-Makrophagen) oder Makrophagen mit natürlich niedriger Expression von MafB oder cMaf, wie z.B. alveoläre Makrophagen, weisen dementsprechend eine erweiterte Kapazität zur Selbsterneuerung auf. Wie haben festgestellt, dass eine kurze Isoform des Maus- Gens Piwil2, die wir ‚Piwito’ genannt haben, in MafDKO und alveolären Makrophagen exprimiert wird. Die Expression von Piwito ist für die normale Selbsterneuerung der untersuchten Makrophagen notwendig, wie die in vitro und in vivo Untersuchungen darlegen. Eine Abwesenheit von Piwito in alveolären Makrophagen führt zu einer Verkürzung derer Lebenspanne in Kultur. Außerdem beweisen wir, dass Piwito von MafB in nicht-proliferierenden Makrophagen gebunden und unterdrückt wird. Diese Studie ist somit der erste Bericht über eine somatische Funktion von PIWI-Proteinen in nicht transformierten Zellen von Säugetieren., PIWI proteins are the main players of an RNA-based gene regulatory machinery that represses transposable elements in the genome to prevent their mobilization and ensure genetic stability. PIWI proteins have thus highly conserved stem-cell functions. They are indispensable for the long-term maintenance of the somatic stem cells that drive regeneration in invertebrates, of various adult somatic stem cells in Drosophila and, most prominently, of the germline of all species studied so far. In mammals, their described functions are strictly restricted to the male germline. Despite suggestive observations for a role of PIWI proteins in the mammalian soma, robust evidence remains absent. Similar to stem cells, tissue macrophages can locally self-renew to maintain their populations. Mechanistically, their self-renewal relies on low expression of the macrophage transcription factors MafB and cMaf, since it allows the induction of a stem cell-like network of genes that drives proliferation. Macrophages with a genetic deletion of MafB and cMaf (MafDKO macrophages) acquire therefore the capacity to self-renew, defined by an indefinite growth in culture that does not comprise their identity and does not involve cancerogenic transformation. Similarly, macrophages with naturally low levels of MafB or cMaf, such as alveolar macrophages, display an extended self-renewal capacity in vivo and in vitro. We have found that a short isoform of the murine Piwil2 gene, that we named ‘Piwito’, is expressed in MafDKO and alveolar macrophages. Piwito expression is necessary for the unaltered self-renewal of macrophages, as shown by in vitro and in vivo assays. To highlight is the fact that Piwito deficiency limits the extended lifespan of alveolar macrophages in culture. Additionally, we show that Piwito is bound and repressed by MafB in quiescent macrophages. This study thus represents the first report of a somatic function for mammalian PIWI proteins in non-transformed cells.

Nach dem rekordwarmen Jahr 2018 war es nur logisch, dass der Vormittag des Rebbautags den Klimawandel zum Thema hatte. Am Nachmittag wurden die wichtigsten Label für nachhaltige Produktion präsentiert. Der Weinbereitungstag war der Vinifikation und Vermarktung der "neuen Rebsorten" gewidmet.

RNA interference is a crucial antiviral mechanism in arthropods, including in mosquito vectors of arthropod-borne viruses (arboviruses). Although the exogenous small interfering RNA (siRNA) pathway constitutes an efficient antiviral response in mosquitoes, virus-derived P-element induced wimpy testis (PIWI)-interacting RNAs (piRNAs) have been implicated in the response to alpha-, bunya- and flaviviruses in Aedes spp. mosquitoes. Culex mosquitoes transmit several medically important viruses including West Nile virus (WNV), but are considerably less well studied than Aedes mosquitoes and little is known about antiviral RNA interference in Culex mosquitoes. Therefore, we sequenced small RNA (sRNA) libraries from different Culex cell lines and tissues infected with WNV. The clear majority of virus-derived sRNA reads were 21 nt siRNAs in all cell lines and tissues tested, with no evidence for a role of WNV-derived piRNAs. Additionally, we aligned sRNA reads from Culex quinquefasciatus Hsu cells to the insect-specific rhabdovirus, Merida virus, which persistently replicates in these cells. We found that a significant proportion of the sRNA response to Merida virus consisted of piRNAs. Since viral DNA forms have been implicated in siRNA and piRNA responses of Aedes spp. mosquitoes, we also tested for viral DNA forms in WNV infected Culex cells. We detected viral DNA in Culex tarsalis cells infected with WNV and, to a lesser amount, WNV and Merida virus-derived DNA in Culex quinquefasciatus Hsu cells. In conclusion, Hsu cells generated Merida virus-derived piRNAs, but our data suggests that the major sRNA response of Culex cells and mosquitoes to WNV infection is the exogenous siRNA response. It is also evident that sRNA responses differ significantly between specific virus-mosquito combinations. Future work using additional Culex-borne viruses may further elucidate how virus-derived piRNAs are generated in Culex cells and what role they may play in controlling replication

Oral presentation

The PIWI-interacting RNA (piRNA) pathway has long been thought to function solely in the germline, but evidence for its functions in somatic cells is emerging. Here we report an unexpected role for the piRNA pathway in Caenorhabditis elegans sensory axon regeneration after injury. Loss of function in a subset of components of the piRNA pathway results in enhanced axon regrowth. Two essential piRNA factors, PRDE-1 and PRG-1/PIWI, inhibit axon regeneration in a gonad-independent and cell-autonomous manner. By smFISH analysis we find that prde-1 transcripts are present in neurons, as well as germ cells. The piRNA pathway inhibits axon regrowth independent of nuclear transcriptional silencing but dependent on the slicer domain of PRG-1/PIWI, suggesting that post-transcriptional gene silencing is involved. Our results reveal the neuronal piRNA pathway as a novel intrinsic repressor of axon regeneration.

Oral presentation

The aim of the research presented in this thesis is to examine the piRNA pathway in Aedes aegypti, with an emphasis on understanding the role of the pathway in the soma. Chapter one reviews the piRNA pathway’s role in transposon regulation as well as transposon-independent roles, such as sex-determination in Bombyx mori. In addition, preliminary research from the Atkinson laboratory showed an expansion in the number and expression domain of the PIWI family in Aedes aegypti compared to a model Dipteran organism, Drosophila melanogaster. Chapter two introduces research I performed that showed the somatic expression of an important PIWI gene, Ago 3, in somatic ovarian follicular cells and larval gastric caecum. Piwi 2 was found to have a germline localization. In addition, an Ago 3 RNAi knockdown line (M14) exhibited a phenotype of larval mortality. Chapter three focuses on a new, more stringent method of annotating piRNA clusters in Ae. aegypti from different types of mosquito sRNA libraries, including both somatic and germline tissue. Two fairly distinct sets of piRNA clusters were discovered, one in the soma and one in the germline. Somatic clusters produced piRNA against predominately gypsy elements; somatic piRNA bore strong U1 signatures but weaker A10 signatures, and also bore less hallmarks of the piRNA ping-pong amplification loop. In contrast, germline clusters produced piRNA against a more varied set of transposons, and germline piRNA had both strong U1 and A10 signatures. Germline libraries also had larger quantities of transposon-derived piRNA. Chapter four examines the effect of Ago 3 knockdown in mosquito larvae. Modest decreases in U1 and A10 signatures were seen in piRNA sequenced from Ago 3 knockdown mosquitoes; in addition, the relative percent of piRNA mapping against transposons declined from wild-type and control conditions. A global decrease in mRNA mapping to transposons was also detected. Together, these data show that somatic piRNAs exist in A

The aim of the research presented in this thesis is to examine the piRNA pathway in Aedes aegypti, with an emphasis on understanding the role of the pathway in the soma. Chapter one reviews the piRNA pathway’s role in transposon regulation as well as transposon-independent roles, such as sex-determination in Bombyx mori. In addition, preliminary research from the Atkinson laboratory showed an expansion in the number and expression domain of the PIWI family in Aedes aegypti compared to a model Dipteran organism, Drosophila melanogaster. Chapter two introduces research I performed that showed the somatic expression of an important PIWI gene, Ago 3, in somatic ovarian follicular cells and larval gastric caecum. Piwi 2 was found to have a germline localization. In addition, an Ago 3 RNAi knockdown line (M14) exhibited a phenotype of larval mortality. Chapter three focuses on a new, more stringent method of annotating piRNA clusters in Ae. aegypti from different types of mosquito sRNA libraries, including both somatic and germline tissue. Two fairly distinct sets of piRNA clusters were discovered, one in the soma and one in the germline. Somatic clusters produced piRNA against predominately gypsy elements; somatic piRNA bore strong U1 signatures but weaker A10 signatures, and also bore less hallmarks of the piRNA ping-pong amplification loop. In contrast, germline clusters produced piRNA against a more varied set of transposons, and germline piRNA had both strong U1 and A10 signatures. Germline libraries also had larger quantities of transposon-derived piRNA. Chapter four examines the effect of Ago 3 knockdown in mosquito larvae. Modest decreases in U1 and A10 signatures were seen in piRNA sequenced from Ago 3 knockdown mosquitoes; in addition, the relative percent of piRNA mapping against transposons declined from wild-type and control conditions. A global decrease in mRNA mapping to transposons was also detected. Together, these data show that somatic piRNAs exist in A

Transgenerational transmission of genome-regulatory epigenetic information can determine phenotypes in the progeny of sexual reproduction. Sequence specificity of transgenerational regulation derives from small RNAs assembled into Piwi-protein complexes. Known targets of transgenerational regulation are primarily transposons and transposon-derived sequences. Here, we extend the scope of Piwi-mediated transgenerational regulation to include unique noncoding RNA loci. Ciliates such as Tetrahymena have a phenotypically silent germline micronucleus and an expressed somatic macronucleus, which is differentiated anew from a germline genome copy in sexual reproduction. We show that the nuclear-localized Tetrahymena Piwi protein Twi8p shuttles from parental to zygotic macronuclei. Genetic elimination of Twi8p has no phenotype for cells in asexual growth. On the other hand, cells lacking Twi8p arrest in sexual reproduction with zygotic nuclei that retain the germline genome structure, without the DNA elimination and fragmentation required to generate a functional macronucleus. Twi8p-bound small RNAs originate from long-noncoding RNAs with a terminal hairpin, which become detectable in the absence of Twi8p. Curiously, the loci that generate Twi8p-bound small RNAs are essential for asexual cell growth, even though Twi8 RNPs are essential only in sexual reproduction. Our findings suggest the model that Twi8 RNPs act on silent germline chromosomes to permit their conversion to expressed macronuclear chromosomes. Overall this work reveals that a Piwi protein carrying small RNAs from long-noncoding RNA loci has transgenerational function in establishing zygotic nucleus competence for gene expression.

Small RNAs are essential for a variety of cellular functions. Argonaute (AGO) proteins are associated with all of the different classes of small RNAs, and are indispensable in small RNA-mediated regulatory pathways. AGO proteins have been identified in various types of stem cells in diverse species from plants and animals. This review article highlights recent progress on how AGO proteins and AGO-bound small RNAs regulate the self-renewal and differentiation of distinct stem cell types, including pluripotent, germline, somatic, and cancer stem cells.

Piwi-interacting RNAs (piRNAs), whose role in germline maintenance has been established, are now also being classified as post-transcriptional regulators of gene expression in somatic cells. PIWI proteins, central to piRNA biogenesis, have been identified as genetic and epigenetic regulators of gene expression. piRNAs/PIWIs have emerged as potential biomarkers for cancer but their relevance to breast cancer has not been comprehensively studied. piRNAs and mRNAs were profiled from normal and breast tumor tissues using next generation sequencing and Agilent platforms, respectively. Gene targets for differentially expressed piRNAs were identified from mRNA expression dataset. piRNAs and PIWI genes were independently assessed for their prognostic significance (outcomes: Overall Survival, OS and Recurrence Free Survival, RFS). We discovered eight piRNAs as novel independent prognostic markers and their association with OS was confirmed in an external dataset (The Cancer Genome Atlas). Further, PIWIL3 and PIWIL4 genes showed prognostic relevance. 306 gene targets exhibited reciprocal relationship with piRNA expression. Cancer cell pathways such as apoptosis and cell signaling were the key Gene Ontology terms associated with the regulated gene targets. Overall, we have captured the entire cascade of events in a dysregulated piRNA pathway and have identified novel markers for breast cancer prognostication.

Piwi-interacting RNAs (piRNAs), whose role in germline maintenance has been established, are now also being classified as post-transcriptional regulators of gene expression in somatic cells. PIWI proteins, central to piRNA biogenesis, have been identified as genetic and epigenetic regulators of gene expression. piRNAs/PIWIs have emerged as potential biomarkers for cancer but their relevance to breast cancer has not been comprehensively studied. piRNAs and mRNAs were profiled from normal and breast tumor tissues using next generation sequencing and Agilent platforms, respectively. Gene targets for differentially expressed piRNAs were identified from mRNA expression dataset. piRNAs and PIWI genes were independently assessed for their prognostic significance (outcomes: Overall Survival, OS and Recurrence Free Survival, RFS). We discovered eight piRNAs as novel independent prognostic markers and their association with OS was confirmed in an external dataset (The Cancer Genome Atlas). Further, PIWIL3 and PIWIL4 genes showed prognostic relevance. 306 gene targets exhibited reciprocal relationship with piRNA expression. Cancer cell pathways such as apoptosis and cell signaling were the key Gene Ontology terms associated with the regulated gene targets. Overall, we have captured the entire cascade of events in a dysregulated piRNA pathway and have identified novel markers for breast cancer prognostication.

Piwi-interacting RNAs (piRNAs), whose role in germline maintenance has been established, are now also being classified as post-transcriptional regulators of gene expression in somatic cells. PIWI proteins, central to piRNA biogenesis, have been identified as genetic and epigenetic regulators of gene expression. piRNAs/PIWIs have emerged as potential biomarkers for cancer but their relevance to breast cancer has not been comprehensively studied. piRNAs and mRNAs were profiled from normal and breast tumor tissues using next generation sequencing and Agilent platforms, respectively. Gene targets for differentially expressed piRNAs were identified from mRNA expression dataset. piRNAs and PIWI genes were independently assessed for their prognostic significance (outcomes: Overall Survival, OS and Recurrence Free Survival, RFS). We discovered eight piRNAs as novel independent prognostic markers and their association with OS was confirmed in an external dataset (The Cancer Genome Atlas). Further, PIWIL3 and PIWIL4 genes showed prognostic relevance. 306 gene targets exhibited reciprocal relationship with piRNA expression. Cancer cell pathways such as apoptosis and cell signaling were the key Gene Ontology terms associated with the regulated gene targets. Overall, we have captured the entire cascade of events in a dysregulated piRNA pathway and have identified novel markers for breast cancer prognostication.

Small RNAs are essential for a variety of cellular functions. Argonaute (AGO) proteins are associated with all of the different classes of small RNAs, and are indispensable in small RNA-mediated regulatory pathways. AGO proteins have been identified in various types of stem cells in diverse species from plants and animals. This review article highlights recent progress on how AGO proteins and AGO-bound small RNAs regulate the self-renewal and differentiation of distinct stem cell types, including pluripotent, germline, somatic, and cancer stem cells.

Piwi-interacting RNAs (piRNAs), whose role in germline maintenance has been established, are now also being classified as post-transcriptional regulators of gene expression in somatic cells. PIWI proteins, central to piRNA biogenesis, have been identified as genetic and epigenetic regulators of gene expression. piRNAs/PIWIs have emerged as potential biomarkers for cancer but their relevance to breast cancer has not been comprehensively studied. piRNAs and mRNAs were profiled from normal and breast tumor tissues using next generation sequencing and Agilent platforms, respectively. Gene targets for differentially expressed piRNAs were identified from mRNA expression dataset. piRNAs and PIWI genes were independently assessed for their prognostic significance (outcomes: Overall Survival, OS and Recurrence Free Survival, RFS). We discovered eight piRNAs as novel independent prognostic markers and their association with OS was confirmed in an external dataset (The Cancer Genome Atlas). Further, PIWIL3 and PIWIL4 genes showed prognostic relevance. 306 gene targets exhibited reciprocal relationship with piRNA expression. Cancer cell pathways such as apoptosis and cell signaling were the key Gene Ontology terms associated with the regulated gene targets. Overall, we have captured the entire cascade of events in a dysregulated piRNA pathway and have identified novel markers for breast cancer prognostication.

Piwi-interacting RNAs (piRNAs), whose role in germline maintenance has been established, are now also being classified as post-transcriptional regulators of gene expression in somatic cells. PIWI proteins, central to piRNA biogenesis, have been identified as genetic and epigenetic regulators of gene expression. piRNAs/PIWIs have emerged as potential biomarkers for cancer but their relevance to breast cancer has not been comprehensively studied. piRNAs and mRNAs were profiled from normal and breast tumor tissues using next generation sequencing and Agilent platforms, respectively. Gene targets for differentially expressed piRNAs were identified from mRNA expression dataset. piRNAs and PIWI genes were independently assessed for their prognostic significance (outcomes: Overall Survival, OS and Recurrence Free Survival, RFS). We discovered eight piRNAs as novel independent prognostic markers and their association with OS was confirmed in an external dataset (The Cancer Genome Atlas). Further, PIWIL3 and PIWIL4 genes showed prognostic relevance. 306 gene targets exhibited reciprocal relationship with piRNA expression. Cancer cell pathways such as apoptosis and cell signaling were the key Gene Ontology terms associated with the regulated gene targets. Overall, we have captured the entire cascade of events in a dysregulated piRNA pathway and have identified novel markers for breast cancer prognostication.

Piwi-interacting RNAs (piRNAs), whose role in germline maintenance has been established, are now also being classified as post-transcriptional regulators of gene expression in somatic cells. PIWI proteins, central to piRNA biogenesis, have been identified as genetic and epigenetic regulators of gene expression. piRNAs/PIWIs have emerged as potential biomarkers for cancer but their relevance to breast cancer has not been comprehensively studied. piRNAs and mRNAs were profiled from normal and breast tumor tissues using next generation sequencing and Agilent platforms, respectively. Gene targets for differentially expressed piRNAs were identified from mRNA expression dataset. piRNAs and PIWI genes were independently assessed for their prognostic significance (outcomes: Overall Survival, OS and Recurrence Free Survival, RFS). We discovered eight piRNAs as novel independent prognostic markers and their association with OS was confirmed in an external dataset (The Cancer Genome Atlas). Further, PIWIL3 and PIWIL4 genes showed prognostic relevance. 306 gene targets exhibited reciprocal relationship with piRNA expression. Cancer cell pathways such as apoptosis and cell signaling were the key Gene Ontology terms associated with the regulated gene targets. Overall, we have captured the entire cascade of events in a dysregulated piRNA pathway and have identified novel markers for breast cancer prognostication.

Gonads are specialized gamete-producing structures that, despite their functional importance, are generated by diverse mechanisms across groups of animals and can be among the most plastic organs of the body. Annelids, the segmented worms, are a group in which gonads have been documented to be plastic and to be able to regenerate, but little is known about what factors influence gonad development or how these structures regenerate. In this study, we aimed to identify factors that influence the presence and size of gonads and to investigate gonad regeneration in the small asexually reproducing annelid, Pristina leidyi. We found that gonad presence and size in asexual adult P. leidyi are highly variable across individuals and identified several factors that influence these structures. An extrinsic factor, food availability, and two intrinsic factors, individual age and parental age, strongly influence the presence and size of gonads in P. leidyi. We also found that following head amputation in this species, gonads can develop by morphallactic regeneration in previously non-gonadal segments. We also identified a sexually mature individual from our laboratory culture that demonstrates that, although our laboratory strain reproduces only asexually, it retains the potential to become fully sexual. Our findings demonstrate that gonads in P. leidyi display high phenotypic plasticity and flexibility with respect to their presence, their size, and the segments in which they can form. Considering our findings along with relevant data from other species, we find that, as a group, clitellate annelids can form gonads in at least four different contexts: post-starvation refeeding, fission, morphallactic regeneration, and epimorphic regeneration. This group is thus particularly useful for investigating the mechanisms involved in gonad formation and the evolution of post-embryonic phenotypic plasticity.

Correct transmission of the genetic material to the next generation is essential for the maintenance of genomic integrity. Therefore chromosomes should be divided properly during each meiotic division. In this thesis the function of several proteins, particularly Transforming acidic coiled-coil protein 3 (TACC3), has been examined in cattle oocytes. Immunofluorescent staining demonstrated the presence of TACC3 around the chromosomes during the first and second meiotic divisions. The function of TACC3 was investigated directly by injecting TACC3 targeted siRNAs into oocytes and indirectly by chemical inhibition of Aurora A activity, a kinase known to phosphorylate and thereby activate TACC3. Abnormal spindle formation and impaired embryonic development was observed. It is concluded that expression of TACC3 is important for the stability of the meiotic spindle and thereby correct segregation of chromosomes. For the survival of a species, it is also important that the genome in the gametes is protected from potentially damaging elements such as transposons. Piwi proteins, together with Piwi-interacting RNAs (piRNAs), can suppress transposon activity by endonuclease activity of the Piwi protein using piRNA as a guide for target recognition. Small RNAs in ovaries and oocytes were sequenced. In cattle ovaries, the pachytene like 30 nucleotide (nt) piRNAs were detected. Using immunohistochemistry, PIWIL1 was demonstrated in the primordial follicles, suggesting that the 30 nt piRNAs in the ovaries interact with PIWIL1. In the ovaries of adult women, expression of PIWIL1 and PIWIL2 was detected, together with piRNAs of 27-28 and 30 nt, whereas in ovaries from second trimester fetuses 27 nt piRNAs were detected, as was PIWIL2 expression. It is proposed that in oocytes from fetal follicles, PIWIL2 interacts with 27 nt piRNAs, whereas in adult ovaries, and particularly the oocytes of primary follicles, PIWIL1 and PIWIL2 function with 27-28 and 30 nt piRNAs. In bovine oocyte

The ability of transposons to mobilize to new places in a genome enables them to introgress rapidly into populations. Transposons have been used as tools in mosquitoes to genetically transform a number of species including Anopheles stephensi, a vector of human malaria. These mobile genetic elements also have been proposed as tools to drive anti-pathogen effector genes into wild mosquito populations to replace pathogen-susceptible insects with those engineered genetically to be resistant to or unable to transmit a pathogen. The piRNA pathway has been characterized recently in the germ-line of the fruit fly, Drosophila melanogaster, and is responsible for down-regulating transposon mobility. In order to use transposons effectively for vector and disease control, we need an understanding of the biology of the interplay between mosquitoes and synthetic transposon constructs. Presented here are: 1) evidence that components of the piRNA pathway are present in An. stephensi and expressed in a manner spatially and temporally appropriate for transposon control; 2) a proof-of-principle for a synthetic autonomous transposon-based construct, showing that exogenous genes can be encoded to self-mobilize; and 3) an investigation into a link between stress, transposon mobility and the piRNA pathway.

Arboviruses cause an overwhelming number of clinical cases of disease each year around the world. The kinetics of viral replication is of critical importance to the dissemination of virus within the mosquito and ultimately transmission between hosts. Arboviral replication and dissemination is dependent upon their ability to evade the immune response and modulate cell toxicity in two separate hosts. Although there is a tremendous amount of study on the replication of arboviruses in mammalian hosts, there is much less focus on their replication in insects. In mosquitos the siRNA pathway of RNA interference (RNAi) is an indispensible component of the antiviral immune system and a key repressor of viral replication. In this work, we explore two poorly understood aspects of antiviral RNAi in mosquitoes: systemic dsRNA spread and piRNA mediated immunity. We show that the Aedes aegypti cells, Aag2, effectively take up long dsRNA from the extracellular medium to initiate RNAi. Pharmacological and genetic analyses reveal that dsRNA enters the cell via clathrin-mediated endocytosis. Uptake of exogenous dsRNA directed against Sindbis virus (SINV) inhibits viral replication. However, SINV inhibits RNAi initiated by dsRNA soaking after infection by inhibiting acidification of endosomes. Thus, Sindbis virus may control RNAi antiviral immunity in mosquitoes by suppressing exogenous dsRNA uptake. In addition to its biological role, from a technical point of view the observation that RNAi can be initiated by naked dsRNA in Aedes aegypti cells may facilitate studies encompassing a wide variety of biological processes in mosquitoes. We also show that in infected cells and mosquitos, both virally derived siRNAs (v-siRNAs) and piRNAs (v-piRNAs) are detected in Aedes aegypti. Although the piRNA pathway is generally associated with germline defense against selfish genetic elements such as transposons, in Aedes aegypti the piRNA pathway mediates antiviral immunity in vivo in somatic tissue

Background Specific genomic loci, termed Piwi-interacting RNA (piRNA) clusters, manufacture piRNAs that serve as guides for the inactivation of complementary transposable elements (TEs). The piRNA pathway has been accurately detailed in Drosophila melanogaster, while it remains poorly examined in other insects. This pathway is increasingly recognized as critical for germline development and reproduction. Understanding of the piRNA functions in mosquitoes could offer an opportunity for disease vector control by the reduction of their reproductive potential. Results To analyze the similarities and differences in this pathway between Drosophila and mosquito, we performed an in-depth analysis of the genomic loci producing piRNAs and their targets in the African malaria vector Anopheles gambiae. We identified 187 piRNA clusters in the An. gambiae genome and 155 piRNA clusters in the D. melanogaster genome. We demonstrate that many more piRNA clusters in the mosquito compared with the fruit fly are uni-directionally transcribed and are located outside pericentromeric heterochromatin. About 11 % of the An. gambiae piRNA population map to gene transcripts. This is a noticeable increase compared with the ~6 % of the piRNA population mapped to genes in D. melanogaster. A subset of the piRNA-enriched genes in An. gambiae has functions related to reproduction and development. At least 24 and 65 % of the mapped piRNAs correspond to genomic TE sequences in An. gambiae and D. melanogaster, respectively. DNA transposons and non-LTR retrotransposons are more abundant in An. gambiae, while LTR retrotransposons are more abundant in D. melanogaster. Yet, piRNAs predominantly target LTR retrotransposons in both species, which may point to a distinct feature of these elements compared to the other classes of TEs concerning their silencing by the piRNA pathway. Conclusions Here, we demonstrate that piRNA-producing loci have more ubiquitous distribution in the An. gambiae genome than in th

The ability of transposons to mobilize to new places in a genome enables them to introgress rapidly into populations. Transposons have been used as tools in mosquitoes to genetically transform a number of species including Anopheles stephensi, a vector of human malaria. These mobile genetic elements also have been proposed as tools to drive anti-pathogen effector genes into wild mosquito populations to replace pathogen-susceptible insects with those engineered genetically to be resistant to or unable to transmit a pathogen. The piRNA pathway has been characterized recently in the germ-line of the fruit fly, Drosophila melanogaster, and is responsible for down-regulating transposon mobility. In order to use transposons effectively for vector and disease control, we need an understanding of the biology of the interplay between mosquitoes and synthetic transposon constructs. Presented here are: 1) evidence that components of the piRNA pathway are present in An. stephensi and expressed in a manner spatially and temporally appropriate for transposon control; 2) a proof-of-principle for a synthetic autonomous transposon-based construct, showing that exogenous genes can be encoded to self-mobilize; and 3) an investigation into a link between stress, transposon mobility and the piRNA pathway.

Arboviruses cause an overwhelming number of clinical cases of disease each year around the world. The kinetics of viral replication is of critical importance to the dissemination of virus within the mosquito and ultimately transmission between hosts. Arboviral replication and dissemination is dependent upon their ability to evade the immune response and modulate cell toxicity in two separate hosts. Although there is a tremendous amount of study on the replication of arboviruses in mammalian hosts, there is much less focus on their replication in insects. In mosquitos the siRNA pathway of RNA interference (RNAi) is an indispensible component of the antiviral immune system and a key repressor of viral replication. In this work, we explore two poorly understood aspects of antiviral RNAi in mosquitoes: systemic dsRNA spread and piRNA mediated immunity. We show that the Aedes aegypti cells, Aag2, effectively take up long dsRNA from the extracellular medium to initiate RNAi. Pharmacological and genetic analyses reveal that dsRNA enters the cell via clathrin-mediated endocytosis. Uptake of exogenous dsRNA directed against Sindbis virus (SINV) inhibits viral replication. However, SINV inhibits RNAi initiated by dsRNA soaking after infection by inhibiting acidification of endosomes. Thus, Sindbis virus may control RNAi antiviral immunity in mosquitoes by suppressing exogenous dsRNA uptake. In addition to its biological role, from a technical point of view the observation that RNAi can be initiated by naked dsRNA in Aedes aegypti cells may facilitate studies encompassing a wide variety of biological processes in mosquitoes. We also show that in infected cells and mosquitos, both virally derived siRNAs (v-siRNAs) and piRNAs (v-piRNAs) are detected in Aedes aegypti. Although the piRNA pathway is generally associated with germline defense against selfish genetic elements such as transposons, in Aedes aegypti the piRNA pathway mediates antiviral immunity in vivo in somatic tissue

Background Specific genomic loci, termed Piwi-interacting RNA (piRNA) clusters, manufacture piRNAs that serve as guides for the inactivation of complementary transposable elements (TEs). The piRNA pathway has been accurately detailed in Drosophila melanogaster, while it remains poorly examined in other insects. This pathway is increasingly recognized as critical for germline development and reproduction. Understanding of the piRNA functions in mosquitoes could offer an opportunity for disease vector control by the reduction of their reproductive potential. Results To analyze the similarities and differences in this pathway between Drosophila and mosquito, we performed an in-depth analysis of the genomic loci producing piRNAs and their targets in the African malaria vector Anopheles gambiae. We identified 187 piRNA clusters in the An. gambiae genome and 155 piRNA clusters in the D. melanogaster genome. We demonstrate that many more piRNA clusters in the mosquito compared with the fruit fly are uni-directionally transcribed and are located outside pericentromeric heterochromatin. About 11 % of the An. gambiae piRNA population map to gene transcripts. This is a noticeable increase compared with the ~6 % of the piRNA population mapped to genes in D. melanogaster. A subset of the piRNA-enriched genes in An. gambiae has functions related to reproduction and development. At least 24 and 65 % of the mapped piRNAs correspond to genomic TE sequences in An. gambiae and D. melanogaster, respectively. DNA transposons and non-LTR retrotransposons are more abundant in An. gambiae, while LTR retrotransposons are more abundant in D. melanogaster. Yet, piRNAs predominantly target LTR retrotransposons in both species, which may point to a distinct feature of these elements compared to the other classes of TEs concerning their silencing by the piRNA pathway. Conclusions Here, we demonstrate that piRNA-producing loci have more ubiquitous distribution in the An. gambiae genome than in th

Background Specific genomic loci, termed Piwi-interacting RNA (piRNA) clusters, manufacture piRNAs that serve as guides for the inactivation of complementary transposable elements (TEs). The piRNA pathway has been accurately detailed in Drosophila melanogaster, while it remains poorly examined in other insects. This pathway is increasingly recognized as critical for germline development and reproduction. Understanding of the piRNA functions in mosquitoes could offer an opportunity for disease vector control by the reduction of their reproductive potential. Results To analyze the similarities and differences in this pathway between Drosophila and mosquito, we performed an in-depth analysis of the genomic loci producing piRNAs and their targets in the African malaria vector Anopheles gambiae. We identified 187 piRNA clusters in the An. gambiae genome and 155 piRNA clusters in the D. melanogaster genome. We demonstrate that many more piRNA clusters in the mosquito compared with the fruit fly are uni-directionally transcribed and are located outside pericentromeric heterochromatin. About 11 % of the An. gambiae piRNA population map to gene transcripts. This is a noticeable increase compared with the ~6 % of the piRNA population mapped to genes in D. melanogaster. A subset of the piRNA-enriched genes in An. gambiae has functions related to reproduction and development. At least 24 and 65 % of the mapped piRNAs correspond to genomic TE sequences in An. gambiae and D. melanogaster, respectively. DNA transposons and non-LTR retrotransposons are more abundant in An. gambiae, while LTR retrotransposons are more abundant in D. melanogaster. Yet, piRNAs predominantly target LTR retrotransposons in both species, which may point to a distinct feature of these elements compared to the other classes of TEs concerning their silencing by the piRNA pathway. Conclusions Here, we demonstrate that piRNA-producing loci have more ubiquitous distribution in the An. gambiae genome than in th

The ability of transposons to mobilize to new places in a genome enables them to introgress rapidly into populations. The piRNA pathway has been characterized recently in the germ line of the fruit fly, Drosophila melanogaster, and is responsible for downregulating transposon mobility. Transposons have been used as tools in mosquitoes to genetically transform a number of species including Anopheles stephensi, a vector of human malaria. These mobile genetic elements also have been proposed as tools to drive antipathogen effector genes into wild mosquito populations to replace pathogen-susceptible insects with those engineered genetically to be resistant to or unable to transmit a pathogen. The piRNA pathway may affect the performance of such proposed genetic engineering strategies. In the present study, we identify and describe the An. stephensi orthologues of the major genes in the piRNA pathway, Ago3, Aubergine (Aub) and Piwi. Consistent with a role in protection from transposon movement, these three genes are expressed constitutively in the germ-line cells of ovaries and induced further after a blood meal.

The ability of transposons to mobilize to new places in a genome enables them to introgress rapidly into populations. The piRNA pathway has been characterized recently in the germ line of the fruit fly, Drosophila melanogaster, and is responsible for downregulating transposon mobility. Transposons have been used as tools in mosquitoes to genetically transform a number of species including Anopheles stephensi, a vector of human malaria. These mobile genetic elements also have been proposed as tools to drive antipathogen effector genes into wild mosquito populations to replace pathogen-susceptible insects with those engineered genetically to be resistant to or unable to transmit a pathogen. The piRNA pathway may affect the performance of such proposed genetic engineering strategies. In the present study, we identify and describe the An. stephensi orthologues of the major genes in the piRNA pathway, Ago3, Aubergine (Aub) and Piwi. Consistent with a role in protection from transposon movement, these three genes are expressed constitutively in the germ-line cells of ovaries and induced further after a blood meal.

Pachytene piRNAs are a class of Piwi-interacting small RNAs abundant in spermatids of the adult mouse testis. They are processed from piRNA primary transcripts by a poorly understood mechanism and, unlike fetal transposon-derived piRNAs, lack complementary targets in the spermatid transcriptome. We report that immunopurified complexes of a conserved piRNA pathway protein Maelstrom (MAEL) are enriched in MIWI (Piwi partner of pachytene piRNAs), Tudor-domain proteins and processing intermediates of pachytene piRNA primary transcripts. We provide evidence of functional significance of these complexes in Mael129 knockout mice that exhibit spermiogenic arrest with acrosome and flagellum malformation. Mael129-null mutant testes possess low levels of piRNAs derived from MAEL-associated piRNA precursors and exhibit reduced translation of numerous spermiogenic mRNAs including those encoding acrosome and flagellum proteins. These translation defects in haploid round spermatids are likely indirect, as neither MAEL nor piRNA precursors associate with polyribosomes, and they may arise from an imbalance between pachytene piRNAs and MIWI.

Piwi-interacting RNAs (piRNAs) are a subset of noncoding RNAs that complex with members of the Argonaute protein family, the Piwi proteins. The Piwi/piRNA pathway is highly conserved in animals and is implicated in spermatogenesis, germline stem cell maintenance, and transposon suppression in the germline. In mammals, the latter occurs via de novo DNA methylation of transposons during male germ cell maturation. Available evidence suggests that the Piwi/piRNA pathway may also be active in somatic cells and cancer cells, but this has been little explored. Cancer cells are known to harbour many epigenetic defects, including the hypermethylation of CpG islands (CGI). The mechanism underlying aberrant CGI methylation, which often silences genes whose inactivation favours tumour growth, remains unknown. CGI hypermethylation occurs against a background of global hypomethylation, in which transposons have the potential to become active. In this thesis, I have tested the hypothesis that the Piwi/piRNA system is active in cancer cells and is responsible for directing the aberrant hypermethylation. This hypothesis predicts that mRNAs derived from genes are accidentally captured by the pathway and targeted for silencing in the same way transposons are in the germline. Survey of normal tissues and cancer cell lines revealed that the human Piwi mRNAs (Hiwi, Hili and Hiwi2) are expressed outside the germline. Hiwi2 was found to be ubiquitously expressed, and the protein was localised to the cytoplasm of MDAMB231 cancer cells. Deep sequencing of the small RNAs bound to Hiwi and Hiwi2 in MDAMB231 revealed a complex population of piRNAs derived from genes, and these piRNAs corresponded to unmethylated, rather than methylated CGIs. A prominent population of piRNAs derived from tRNAs was also identified. Mass spectrometry and polysome profiling demonstrated that both Hiwi and Hiwi2 associate with components of the translational machinery, along with other partner proteins known to bind

This thesis examines the mechanisms of class II DNA transposon activity and the genomic defense system against transposons in two Dipteran systems, Drosophila melanogaster and Aedes aegypti. The first half of the dissertation focuses on the affects specific amino acid changes have on target site specificity and transposition activity. Specifically it looks at AeBuster1 and piggyBac mutants. None of the Aebuster1 mutants were found to have increased transposition activity however several mutants had altered target site specificity. One piggyBac mutant hyPBase, which has been shown to have increased activity in HeLA cells, mouse somatic cells and yeast, was studied in-depth. My study determined that hyPBase does not maintain its increased activity in Dipteran systems and significantly increases sterility rates when compared to wild type piggyBac. The second half of the thesis focuses on investigating the piRNA system in the important disease vector Ae. aegypti. The piRNA pathway is a Dicer independent small RNA pathway believed to be responsible for transposon control. In this study I examined the expression of 7 PIWI proteins and studied three of them, AGO3, PIWI2 and PIWI7 using co-immunoprecipitation and RNAi knockdown. Together all of these studies provide insight into transposition mechanisms and transposon control in D. melanogaster and Ae. aegypti.

Small RNA (sRNA)-mediated silencing is a strategy used by almost all eukaryotes to regulate gene expression. The only component common to all sRNA-mediated silencing pathways is a PAZ, PIWI domain-containing (PPD) protein, more commonly called Argonaute/Piwi (Ago/Piwi), after the founding members of the family, which have the same domain structure but fall into two subfamilies based on primary sequence similarity. The conserved PAZ and PIWI domains are required for binding the 3' and 5' ends of the sRNA, respectively. The ribonucleoprotein (RNP) complex then sequence-specifically targets nucleic acid substrates through base pairing between the sRNA guide and the target. Argonaute subfamily proteins and bound sRNAs are ubiquitously expressed in many organisms and have been intensely studied over the past decade. Several mechanisms used by Argonuates to silence gene expression are known in detail. Piwi subfamily proteins and bound RNAs on the other hand are germline-restricted in many organisms and have been intensely studied only for the last five years. They are known to be important for silencing transposons, but detailed mechanisms of their action and other roles are still unknown. Tetrahymena thermophila expresses eight distinct Piwi-subfamily proteins, no Argonaute subfamily proteins, and no transposons, allowing simplified discovery of other Piwi-bound sRNA classes. I have used protein tagging and affinity purification approaches along with deep sequencing to characterize sRNAs bound to each of these Tetrahymena Piwis (Twis), and have uncovered a great diversity of sRNA classes in this organism (Chapter One). In particular, the only essential Piwi in Tetrahymena, Twi12, specifically binds tRNA fragments (Chapter Two). My further studies of Twi12 have revealed its association with the 5' to 3' exoribonuclease Xrn2 in the nucleus (Chapter Three). In the final chapter I describe biochemical techniques and approaches useful for studies of RNPs in Tetrahymena (Chapt

This thesis examines the mechanisms of class II DNA transposon activity and the genomic defense system against transposons in two Dipteran systems, Drosophila melanogaster and Aedes aegypti. The first half of the dissertation focuses on the affects specific amino acid changes have on target site specificity and transposition activity. Specifically it looks at AeBuster1 and piggyBac mutants. None of the Aebuster1 mutants were found to have increased transposition activity however several mutants had altered target site specificity. One piggyBac mutant hyPBase, which has been shown to have increased activity in HeLA cells, mouse somatic cells and yeast, was studied in-depth. My study determined that hyPBase does not maintain its increased activity in Dipteran systems and significantly increases sterility rates when compared to wild type piggyBac. The second half of the thesis focuses on investigating the piRNA system in the important disease vector Ae. aegypti. The piRNA pathway is a Dicer independent small RNA pathway believed to be responsible for transposon control. In this study I examined the expression of 7 PIWI proteins and studied three of them, AGO3, PIWI2 and PIWI7 using co-immunoprecipitation and RNAi knockdown. Together all of these studies provide insight into transposition mechanisms and transposon control in D. melanogaster and Ae. aegypti.