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To align with the target of reducing plant protection measures within the EU Green Deal programme, the utilisation of resistant grape varieties in viticulture becomes indispensable. Consequently, this study investigated the factors that influence consumer acceptance of fungus-resistant grape varieties (FRGV) in Germany. The qualitative research methodology involved conducting semi-structured interviews, including sensory evaluation tests, with focus groups consisting of 48 consumers and evaluating the data using content analysis. The findings revealed that the attractiveness of a grape variety is determined by four distinct attributes: familiarity, designation, association, and image. Furthermore, FRGV can be made more attractive to consumers by using denominations based on established grape varieties or by using and associating them with their sensory patterns. The sensory acceptance of FRGV varies significantly across consumer segments and vinification methods. Providing relevant information about the benefits of resistant grape varieties has a positive impact on consumer acceptance, and the level of interest and relevance varies by target group. The study suggests that it is possible to enhance consumer acceptance by offering attractive grape varieties, targeting group-specific sensory profiles, and engaging in storytelling campaigns that educate consumers about the advantages of resistant grapes.

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.

The piRNA pathway has persisted throughout evolution as an essential regulatory pathway to protect genomic integrity in the metazoan germline. It achieves this through the repression of transposable elements, or “selfish genes,” which would otherwise jump throughout the genome unchecked, causing genomic instability and infertility. While transposable elements are generally deleterious in nature, their persistence in our genome remains an important driver of evolution, both as an agent of mutation and source of raw genetic material. Thus, a delicate balance must be struck to both maintain genomic integrity for the next generation but still enable enough mutation to allow for adaptation. The arms race between the ever-adapting piRNA pathway and its transposon targets provides this balance, and for a long time the piRNA pathway was considered to be germline specific, since that is where both transposon and piRNA pathway activity is highest. It has since become clear that the piRNA pathway is also active in somatic tissue of several invertebrate species, and may even target host genes in some. Whether the piRNA pathway plays a role outside of the germline in vertebrates, however, has remained elusive. In this thesis, we demonstrate that the piRNA pathway is active in a vertebrate somatic cell type, the chick neural crest, where it has been co-opted into the gene regulatory network to repress the transposon-derived gene, ERNI. ERNI, in turn, supresses Sox2 when piRNA pathway protein Piwil1 is downregulated upon neural crest specification. Thus, the piRNA pathway functions to maintain Sox2 expression in the neural plate border stem cell niche, protecting its proliferative abilities and setting the timing of neural crest specification. We also provide preliminary evidence that the neural crest piRNA pathway might be conserved in other vertebrate species, and that two highly conserved transcription factors regulate its expression in the chick neural crest. Our work provides mechanistic insight into a novel function of the piRNA pathway as a regulator of somatic development in vertebrates, and raises the possibility that this ancient pathway may play a more significant role in evolution and transposon co-option by host genomes than previously thought.

As a currently identified small non-coding RNAs (ncRNAs) category, the PIWI-interacting RNAs (piRNAs) are crucial mediators of cell biology. The human genome comprises over 30.000 piRNA genes. Although considered a new field in cancer research, the piRNA pathway is shown by the existing evidence as an active pathway in a variety of different types of cancers with critical impacts on main aspects of cancer progression. Among the regulatory molecules that contribute to maintaining the dynamics of cancer cells, the P-element Induced WImpy testis (PIWI) proteins and piRNAs, as new players, have not been broadly studied so far. Therefore, the identification of cancer-related piRNAs and the assessment of target genes of piRNAs may lead to better cancer prevention and therapy strategies. This review articleaimed to highlight the role and function of piRNAs based on existing data. Understanding the role of piRNA in cancer may provide perspectives on their applications as particular biomarker signature in diagnosis in early stage, prognosis and therapeutic strategies.

8 p.-3 fig.-3 tab., 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., This study was supported by the Agencia Estatal de Investigación; Ministerio de Ciencia, Innovación y Universidades (BFU2017-87095-R), Spain. Miguel A. Brieño-Enriquez was supported by Eunice Kennedy Shriver National Institute of Child Health and Human Development (R00HD090289) and the Magee Auxiliary Research Scholar (MARS) endowment.

PIWI-interacting RNAs (piRNAs) are small RNAs bound by PIWI-clade Argonaute proteins that function to silence transposable elements (TEs). Following mouse Primordial Germ Cell (mPGC) specification around E6.25, fetal piRNAs subsequently emerge in male gonocytes from E13.5 onwards. The in vitro differentiation of mPGC-Like Cells (mPGCLCs) from mESCs has raised the tantalizing prospect of studying the fetal piRNA pathway in greater depth. However, using single-cell RNA-seq and RT-qPCR along mPGCLC differentiation, we find that piRNA pathway factors are not yet expressed in D6 mPGCLCs. Moreover, we do not detect piRNAs across a panel of D6 mPGCLC lines using small RNA-seq. Our combined efforts from two laboratories highlight that in vitro differentiated D6 mPGCLCs do not yet resemble E13.5 or later mouse gonocytes where the piRNA pathway is active, in contrast to a prior report.

Piwi-interacting RNAs (piRNAs) are a novel type of small noncoding RNAs, which are 26–30 nt in length and bind to Piwi proteins. These short RNAs were originally discovered in germline cells and are considered as key regulators for germline maintenance. A growing body of evidence has now extended our views into piRNA biological significance showing that they can also regulate gene expression in somatic cells through transposon silencing, epigenetic programming, DNA rearrangements, mRNA turnover, and translational control. Mounting studies have revealed that the dysregulation of piRNAs may cause epigenetic changes and contribute to diverse diseases. This review illustrates piRNA biogenesis, mechanisms behind piRNA-mediated gene regulation, and changes of piRNAs in different diseases, especially in cancers., Graphical Abstract, Shu and colleagues demonstrate the biogenesis, functions, changes, and probable roles of piRNAs in diseases of different systems, especially in cancers. piRNAs are promising novel biomarkers for early diagnosis and therapeutic targets for precise medicine.

Transposable elements (TEs) comprise a major fraction of vertebrate genomes, yet little is known about their expression and regulation across tissues, and how this varies across major vertebrate lineages. We present the first comparative analysis integrating TE expression and TE regulatory pathway activity in somatic and gametic tissues for a diverse set of 12 vertebrates. We conduct simultaneous gene and TE expression analyses to characterize patterns of TE expression and TE regulation across vertebrates and examine relationships between these features. We find remarkable variation in the expression of genes involved in TE negative regulation across tissues and species, yet consistently high expression in germline tissues, particularly in testes. Most vertebrates show comparably high levels of TE regulatory pathway activity across gonadal tissues except for mammals, where reduced activity of TE regulatory pathways in ovarian tissues may be the result of lower relative germ cell densities. We also find that all vertebrate lineages examined exhibit remarkably high levels of TE-derived transcripts in somatic and gametic tissues, with recently active TE families showing higher expression in gametic tissues. Although most TE-derived transcripts originate from inactive ancient TE families (and are likely incapable of transposition), such high levels of TE-derived RNA in the cytoplasm may have secondary, unappreciated biological relevance.

Membraneless granules assemble in different cell types and cellular loci and are the focus of intense research due to their fundamental importance for cellular organization. These dynamic organelles are commonly assembled from RNA and protein components and exhibit soft matter characteristics of molecular condensates currently characterized with biophysical approaches and super-resolution microscopy imaging. In addition, research on the molecular mechanisms of the RNA–protein granules assembly provided insights into the formation of abnormal granules and molecular aggregates, which takes place during many neurodegenerative disorders including Parkinson’s diseases (PD), Alzheimer’s disease (AD), amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD). While these disorders are associated with formation of abnormal granules, membraneless organelles are normally assembled in neurons and contribute to translational control and affect stability of neuronal RNAs. More recently, a new subtype of membraneless granules was identified in Drosophila glia (glial granules). Interestingly, glial granules were found to contain proteins which are the principal components of the membraneless granules in germ cells (germ granules), indicating some similarity in the functional assembly of these structures in glia and germline. This mini review highlights recent research on glial granules in the context of other membraneless organelles, including their assembly mechanisms and potential functions in the nervous system.

International audience; Piwi-bound small RNAs induce programmed DNA elimination in the ciliated protozoan Tetrahymena. Using the phenomenon called codeletion, this process can be reprogrammed to induce ectopic DNA elimination at basically any given genomic location. Here, we describe the usage of codeletion for genetic studies in Tetrahymena and for investigations of the molecular mechanism of Piwi-directed programmed DNA elimination.

Insertions of transposable elements (TEs) in eukaryotic genomes are usually associated with repressive chromatin, which spreads to neighbouring genomic sequences. In ovaries of Drosophila melanogaster, the Piwi-piRNA pathway plays a key role in the transcriptional silencing of TEs considered to be exerted mostly through the establishment of H3K9me3 histone marks recruiting Heterochromatin Protein 1a (HP1a). Here, using RNA-seq, we investigated the expression of TEs and the adjacent genomic regions upon Piwi and HP1a germline knockdowns sharing a similar genetic background. We found that the depletion of Piwi and HP1a led to the derepression of only partially overlapping TE sets. Several TEs were silenced predominantly by HP1a, whereas the upregulation of some other TEs was more pronounced upon Piwi knockdown and, surprisingly, was diminished upon a Piwi/HP1a double-knockdown. We revealed that HP1a loss influenced the expression of thousands of protein-coding genes mostly not adjacent to TE insertions and, in particular, downregulated a putative transcriptional factor required for TE activation. Nevertheless, our results indicate that Piwi and HP1a cooperatively exert repressive effects on the transcription of euchromatic loci flanking the insertions of some Piwi-regulated TEs. We suggest that this mechanism controls the silencing of a small set of TE-adjacent tissue-specific genes, preventing their inappropriate expression in ovaries.

The PIWI-interacting RNA (piRNA) pathway provides an RNA interference (RNAi) mechanism known from Drosophila studies to maintain the integrity of the germline genome by silencing transposable elements (TE). Aedes aegypti mosquitoes, which are the key vectors of several arthropod-borne viruses, exhibit an expanded repertoire of Piwi proteins involved in the piRNA pathway, suggesting functional divergence. Here, we investigate RNA-binding dynamics and subcellular localization of A. aegypti Piwi4 (AePiwi4), a Piwi protein involved in antiviral immunity and embryonic development, to better understand its function. We found that AePiwi4 PAZ (Piwi/Argonaute/Zwille), the domain that binds the 3′ ends of piRNAs, bound to mature (3′ 2′ O-methylated) and unmethylated RNAs with similar micromolar affinities (KD = 1.7 ± 0.8 μM and KD of 5.0 ± 2.2 μM, respectively; p = 0.05) in a sequence independent manner. Through site-directed mutagenesis studies, we identified highly conserved residues involved in RNA binding and found that subtle changes in the amino acids flanking the binding pocket across PAZ proteins have significant impacts on binding behaviors, likely by impacting the protein secondary structure. We also analyzed AePiwi4 subcellular localization in mosquito tissues. We found that the protein is both cytoplasmic and nuclear, and we identified an AePiwi4 nuclear localization signal (NLS) in the N-terminal region of the protein. Taken together, these studies provide insights on the dynamic role of AePiwi4 in RNAi and pave the way for future studies aimed at understanding Piwi interactions with diverse RNA populations.

RNA interference plays a major role in biology of ciliates. Diverse small RNAs regulate many processes in vegetative cells of ciliates Tetrahymena and Paramecium. Different types of endogenous and exogenous nucleotide sequences induce different RNAi pathways resulting in silencing of the homologous sequences in the macronuclear genome. Likely this way ciliates are able to quickly inactivate heterogeneous sequences and to adapt efficiently to the environmental conditions and external stimuli.

Background. Experimental conditions allow to determine the structural and functional changes of polytene chromosomes under the influence of free ions of an individual metal. Materials and methods. C. plumosus (L.) and G. glaucus (Mg) larvae were placed in solutions of lead nitrate: 0.01, 0.02, 0.1 and 0.5 mg/l. Exposure 12 h. Analysis of preparations of polytene chromosomes was carried out using the Carl Zeiss PrimoStar microscope. The functional activity factor of the nucleolus organizer (NOR), the coefficient of genetic activity of the Balbiani ring (BRR) was calculated. Results. Equations of the dependence of the change in the coefficients: NOR = 5,1870,01 lnC for C. plumosus and NOR = 2,110,03 lnC for G. glaucus; BRR = 1,5040,04 lnC for C. plumosus and BRR = 2,018 + 0,03 lnC for G. glaucus. Conclusion. With an increase in the concentration of lead in both C. plumosus and G. glaucus decreases NOR, which implies a decrease in the intensity of protein biosynthesis processes. BRR decreases in C. plumosus and increases in G. glaucus. The different genome reactions of the two species indicate the existence of different mechanisms of adaptation to lead ions

Made available in DSpace on 2022-04-28T19:47:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2022-02-01 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) ASCRS Research Foundation PiRNAs are a class of small noncoding RNAs that, in their mature form, bind to Piwi proteins to repress transposable element activity. Besides their role in gametogenesis and genome integrity, recent evidence indicates their action in non-germinative tissues. We performed a global analysis of piRNA and Piwi gene expression in the skeletal muscle of juveniles pacu (Piaractus mesopotamicus), tambaqui (Colossoma macropomum), and the hybrid tambacu to evaluate the degree of piRNA sharing among these three genotypes. Total RNA was sequenced and analyzed using specific parameters of piRNAs by bioinformatics tools. piRNA and Piwi gene expression was analyzed by RT-qPCR. We detected 24 piRNA clusters common to the three genotypes, with eight shared between pacu and tambacu, three between pacu and tambaqui, and five between tambaqui and tambacu; seven, five, and four clusters were unique to pacu, tambacu, and tambaqui, respectively. Genomic localization and fold change values showed two clusters and 100 piRNAs shared among the three genotypes. The gene expression of four piRNAs was evaluated to validate our bioinformatics results. piRNAs from cluster 17 were higher in tambacu than pacu and piRNAs from cluster 18 were more highly expressed in tambacu than tambaqui and pacu. In addition, the expression of Piwis 1 and 2 was higher in tambacu and tambaqui than pacu. Our results open an important window to investigate whether these small noncoding RNAs benefit the hybrid in terms of faster growth and offer a new perspective on the function of piRNAs and Piwis in fish skeletal muscle. Department of Structural and Functional Biology Institute of Biosciences Sao Paulo State University – UNESP University of Western São Paulo (UNOESTE), Presidente Prudente Department of Inmmunovirology Faculty of Medicine University of Antioquia Department of Structural and Functional Biology Institute of Biosciences Sao Paulo State University – UNESP ASCRS Research Foundation: 2016/05009-4 ASCRS Research Foundation: 2017/16479-4 CNPq: 307138/2018-6

Vasa, PIWI and TDRKH are conserved components of germ granules that in metazoans are involved in germline specification and differentiation, as documented by mutational experiments in some model animals. So far, investigations on PIWI during spermatogenesis of fish has been limited to a few species, and no information is available for TDRKH, another protein involved in the piRNA pathway. In this study, the immunolocalization of these three germline determinants was analyzed in male gonads of the teleost fish Poecilia reticulata to document their localization pattern in the different stages of germ cell differentiation. To analyze their distribution pattern during the different stages of spermatogenesis we performed immunohistochemistry (IHC) and immunofluorescence (IF) assays using primary polyclonal antibodies after testing their specificity with Western Blot. Moreover, sections of testis stained with haematoxylin and eosin clarified the structural organization of P. reticulata testis, while the use of the confocal microscope and the nuclear staining clarified the different stages of germ cell differentiation during spermatogenesis. The results showed that Vasa, PIWI and TDRKH were specifically immunolocalized in the germ cells of P. reticulata, with no specific signal detected in Sertoli cells and in other somatic cells of the gonad. These markers were detected in all stages of differentiation from early spermatogonia to advanced spermatids. Vasa staining was the strongest in spermatogonia, and then decreases throughout differentiation. Instead, both PIWI and TDRKH staining increases during differentiation, and their distribution pattern, similar to what observed in the mouse, suggests their concerted participation in the piRNA pathway also in this fish.

Planarians are flatworms capable of whole-body regeneration, able to regrow any missing body part after injury or amputation. The extraordinary regenerative capacity of planarians is based upon the presence in the adult of a large population of somatic pluripotent stem cells. These cells, called neoblasts, offer a unique system to study the process of stem cell specification and differentiation in vivo. In recent years, FACS-based isolation of neoblasts, RNAi functional analyses as well as high-throughput approaches such as single-cell sequencing have allowed a rapid progress in our understanding of many different aspects of neoblast biology. Here, we summarize our current knowledge on the molecular signatures that define planarian neoblasts heterogeneity, which includes a percentage of truly pluripotent stem cells, and guide the commitment of pluripotent neoblasts into lineage-specific progenitor cells, as well as their differentiation into specific planarian cell types.

Mesodermal cells of holothurian Eupentacta fraudatrix can transdifferentiate into enterocytes during the regeneration of the digestive system. In this study, we investigated the expression of several genes involved in gut regeneration in E. fraudatrix. Moreover, the localization of progenitor cells of coelomocytes, juvenile cells, and their participation in the formation of the luminal epithelium of the digestive tube were studied. It was shown that Piwi-positive cells were not involved in the formation of the luminal epithelium of the digestive tube. Ef-72 kDa type IV collagenase and Ef-MMP16 had an individual expression profile and possibly different functions. The Ef-tensilin3 gene exhibited the highest expression and indicates its potential role in regeneration. Ef-Sox9/10 and Ef-Sox17 in E. fraudatrix may participate in the mechanism of transdifferentiation of coelomic epithelial cells. Their transcripts mark the cells that plunge into the connective tissue of the gut anlage and give rise to enterocytes. Ef-Sox9/10 probably controls the switching of mesodermal cells to the enterocyte phenotype, while Ef-Sox17 may be involved in the regulation of the initial stages of transdifferentiation.

Mesodermal cells of holothurian Eupentacta fraudatrix can transdifferentiate into enterocytes during the regeneration of the digestive system. In this study, we investigated the expression of several genes involved in gut regeneration in E. fraudatrix. Moreover, the localization of progenitor cells of coelomocytes, juvenile cells, and their participation in the formation of the luminal epithelium of the digestive tube were studied. It was shown that Piwi-positive cells were not involved in the formation of the luminal epithelium of the digestive tube. Ef-72 kDa type IV collagenase and Ef-MMP16 had an individual expression profile and possibly different functions. The Ef-tensilin3 gene exhibited the highest expression and indicates its potential role in regeneration. Ef-Sox9/10 and Ef-Sox17 in E. fraudatrix may participate in the mechanism of transdifferentiation of coelomic epithelial cells. Their transcripts mark the cells that plunge into the connective tissue of the gut anlage and give rise to enterocytes. Ef-Sox9/10 probably controls the switching of mesodermal cells to the enterocyte phenotype, while Ef-Sox17 may be involved in the regulation of the initial stages of transdifferentiation.

P-element-induced wimpy testis (PIWI)-interacting RNAs (piRNAs) are regulatory small non-coding RNAs that participate in transposon inactivation, chromatin regulation, and endogenous gene regulation. Numerous genetic and epigenetic factors regulate cell proliferation and tumor metastasis. PIWI proteins and piRNAs have been revealed to function in regulating upstream or downstream of oncogenes or tumor-suppressor genes in cancer tissues. In the present review, we summarize major recent findings in uncovering the regulation and role of PIWI proteins and piRNAs in tumorigenesis and highlight some of the promising applications of specific piRNAs in cancer therapeutics and as cancer biomarkers.

Objective: Evaluate the messenger RNAs (mRNA) PIWI proteins expression during infection with VDEN 4 in human hepatocyte cells. Materials and Methods: VDEN4 strain H778494 (JQ513335) was used, which was stored in Aedes albopictus cell culture (Clone C6 / 36) at the Arbovirology and Hemorrhagic Fevers section of the Evandro Chagas Institute. The techniques of cell cultures, stock (C6/36), inoculation, extraction, viral load quantification, RTqPCR and statistical analyzes were performed at the Viral Biogenesis Laboratory. Results: According to the results obtained, two cells (HepG2 and Huh7.5) demonstrated a higher level of viral replication at 72 hours post infection (hpi). The PIWI 2 target alter its mRNA expression during VDEN4 infection. The PIWI 4 target expression, was observed an altered expression in the infected cells. Thus, it was found that they are in different poles, while the viral load in the first three days showed high expression. The expression of mRNA was low in relation to the normal rate given by the uninfected cells. Conclusion: In our findings, it was observed that PIWI2 and 4 proteins have an inverse relationship to the viral infection process by VDEN4, when there is an increase in viral replication, these two proteins end up having a significant reduction in their expression. Probably, this reduction of expression is involved with the biogenesis process of apoptosis-regulating microRNAs. Objetivo: Evaluar la expresión de los ARN mensajeros (ARNm) de las proteínas PIWI durante la infección por VDEN 4 en células de hepatocitos humanos. Materiales y Métodos: Se utilizó la cepa VDEN4 H778494 (JQ513335), la cual se almacenó en cultivo celular de Aedes albopictus (Clon C6 / 36) en la colección de la sección de Arbovirología y Fiebres Hemorrágicas del Instituto Evandro Chagas. Las técnicas de cultivo celular, stock (C6/36), inoculación, extracción, cuantificación de carga viral, RTqPCR y análisis estadísticos se realizaron en el Laboratorio de Biogénesis Viral de la misma sección. Resultados: De acuerdo con los resultados obtenidos, se observó que dos células (HepG2 and Huh7.5), demostraron un mayor nivel de replicación viral a las 72 horas post infección (hpi). Se ha demostrado que la diana PIWI 2 altera su expresión de RNAm durante la infección por VDEN4. En la expresión de la diana PIWI 4, se observó una expresión alterada en las células infectadas. Así, se encontró que se encuentran en polos diferentes, mientras que el título viral en los primeros tres días mostró alta expresión, la expresión de RNAm fue baja en relación a la tasa normal dada por la muestra no infectada. Conclusión: en nuestros hallazgos se observó que las proteínas PIWI2 y 4 tienen una relación inversa con el proceso de infección viral por VDEN4, es decir, cuando hay un aumento en la replicación viral, estas proteínas terminan teniendo una reducción significativa en su expresión. . Estos datos terminan por llevarnos a considerar que esta reducción de expresión está involucrada en el proceso de biogénesis de los microRNA reguladores de la apoptosis. Objetivo: Avaliar a expressão dos RNAs mensageiros (mRNA) das proteínas PIWI durante a infecção por VDEN 4 em células de hepatócitos humano. Materiais e Métodos: Foi utilizada a cepa H778494 (JQ513335) do VDEN4 que se encontrava estocada em cultura de células Aedes albopictus (Clone C6/36) no acervo da seção de Arbovirologia e Febres Hemorrágicas do Instituto Evandro Chagas. As técnicas de cultivos celulares (C6/36, HepG2 e Huh7.5), estoque (C6/36), inoculação, extração, quantificação de carga viral, RTqPCR e análises estatísticas foram todas realizadas no Laboratório de Biogênese Viral da mesma seção. Resultados: De acordo com os resultados obtidos observou-se que duas células HepG2 e Huh7.5) demonstraram um maior nível de replicação viral a 72 horas pós infecção (hpi). O alvo PIWI 2 demonstrou que altera sua expressão de mRNA durante a infecção por VDEN4. Na expressão do alvo PIWI 4 foi observado uma expressão alterada nas células infectadas. Assim, verificou-se que os mesmos se encontram em polos diferentes, enquanto o titulo viral nos três primeiros dias apresentaram alta expressão, a expressão de mRNA foi baixa em relação a taxa normal dada pela amostra não infectada. Conclusão: Em nossos achados observou-se que as proteínas PIWI2 e 4 possuem uma relação inversa ao processo de infecção viral por VDEN4, isto é, quando ocorre um aumento da replicação viral essas duas proteínas acabam por ter uma redução significante de sua expressão. Estes dados acabam por nos levar a considerar que esta redução da expressão está envolvida com o processo de biogênese de microRNAs reguladores de apoptose.

Small RNAs are essential for the coordination of many cellular processes, including the regulation of gene expression patterns, the prevention of genomic instability, and the suppression of mutagenic transposon activity. These processes determine aging, longevity, and sensitivity of cells and an organism to stress factors (particularly, ionizing radiation). The biogenesis and activity of small RNAs are provided by proteins of the Argonaute family. These proteins participate in the processing of small RNA precursors and the formation of an RNA-induced silencing complex. However, the role of Argonaute proteins in the regulation of lifespan and radioresistance remains poorly explored. We studied the effect of knockdown of Argonaute genes (AGO1, AGO2, AGO3, piwi) in various tissues on the Drosophila melanogaster lifespan and survival after the γ-irradiation at a dose of 700 Gy. In most cases, these parameters were reduced or did not change significantly in flies with tissue-specific RNA interference. Surprisingly, piwi knockdown in both the fat body and the nervous system caused a lifespan increase. But changes in radioresistance depended on the tissue in which the gene was knocked out. In addition, analysis of changes in retrotransposon levels and expression of stress response genes allowed us to determine associated molecular mechanisms.

Small noncoding RNA pathways have been implicated in diverse mechanisms of gene regulation. In Drosophila ovaries, Piwi binds to Piwi-interacting RNAs (piRNAs) of mostly 24–28 nucleotides (nt) and plays an important role in germline stem cell maintenance, transposon repression, and epigenetic regulation. To understand the mechanism underlying these functions, we report the application of the DamID-seq method to identify genome-wide binding sites of Piwi in Drosophila ovaries. Piwi localizes to at least 4535 euchromatic regions that are enriched with piRNA target sites. Surprisingly, the density of Piwi binding to euchromatin is much higher than in heterochromatin. Disrupting the piRNA binding of Piwi results in an overall change of the genomic binding profile, which indicates the role of piRNAs in directing Piwi to specific genomic sites. Most Piwi binding sites were either within or in the vicinity of protein-coding genes, particularly enriched near the transcriptional start and termination sites. The methylation signal near the transcriptional termination sites is significantly reduced when Piwi was mutated to become defective in piRNA binding. These observations indicate that Piwi might directly regulate the expression of many protein-coding genes, especially through regulating the 3' ends of targeted transcripts.

Tese de mestrado, Biologia Molecular e Genética, Universidade de Lisboa, Faculdade de Ciências, 2021 Submitted by Teresa Boa (tdboa@fc.ul.pt) on 2021-06-26T14:02:38Z No. of bitstreams: 1 TM_Ywen_ZHeng.pdf: 4441887 bytes, checksum: 97f8c69e556cd67923d2e28b8aba3357 (MD5) Made available in DSpace on 2021-06-26T14:02:47Z (GMT). No. of bitstreams: 1 TM_Ywen_ZHeng.pdf: 4441887 bytes, checksum: 97f8c69e556cd67923d2e28b8aba3357 (MD5) Previous issue date: 2021

The transposon silencer piwi genes play important roles in germline determination and maintenance, gametogenesis, and stem-cell self-renewal, and the expression of certain piwi genes is indispensable for regeneration. Knowledge about piwi genes is needed for phylum Nemertea, which contains members (e.g., Lineus sanguineus) with formidable regeneration capacity. By searching the L. sanguineus genome, we identified six Argonaute genes including three ago (Ls-Ago2, Ls-Ago2a, and Ls-Ago2b) and three piwi (Ls-piwi1, Ls-piwi2, and Ls-piwi3) genes. In situ hybridization revealed that, in intact females, Ls-piwi2 and Ls-piwi3 were not expressed, while Ls-piwi1 was expressed in ovaries. During regeneration, Ls-piwi1 and Ls-pcna (proliferating cell nuclear antigen) had strong and similar expressions. The expression of Ls-piwi1 became indetectable while Ls-pcna continued to be expressed when the differentiation of new organs was finished. During anterior regeneration, expression signals of Ls-piwi2 and Ls-piwi3 were weak and only detected in the blastema stage. During posterior regeneration, no expression was observed for Ls-piwi2. To date, no direct evidence has been found for the existence of congenital stem cells in adult L. sanguineus. The &ldquo, pluripotent cells&rdquo, in regenerating tissues are likely to be dedifferentiated from other type(s) of cells.

The transposon silencer piwi genes play important roles in germline determination and maintenance, gametogenesis, and stem-cell self-renewal, and the expression of certain piwi genes is indispensable for regeneration. Knowledge about piwi genes is needed for phylum Nemertea, which contains members (e.g., Lineus sanguineus) with formidable regeneration capacity. By searching the L. sanguineus genome, we identified six Argonaute genes including three ago (Ls-Ago2, Ls-Ago2a, and Ls-Ago2b) and three piwi (Ls-piwi1, Ls-piwi2, and Ls-piwi3) genes. In situ hybridization revealed that, in intact females, Ls-piwi2 and Ls-piwi3 were not expressed, while Ls-piwi1 was expressed in ovaries. During regeneration, Ls-piwi1 and Ls-pcna (proliferating cell nuclear antigen) had strong and similar expressions. The expression of Ls-piwi1 became indetectable while Ls-pcna continued to be expressed when the differentiation of new organs was finished. During anterior regeneration, expression signals of Ls-piwi2 and Ls-piwi3 were weak and only detected in the blastema stage. During posterior regeneration, no expression was observed for Ls-piwi2. To date, no direct evidence has been found for the existence of congenital stem cells in adult L. sanguineus. The “pluripotent cells” in regenerating tissues are likely to be dedifferentiated from other type(s) of cells.

A large fraction of eukaryotic genomes consists of mobile, repetitive elements called transposons. Since their uncontrolled mobilisation is a potentially harmful event, several molecular mechanisms have evolved to counteract transposon activation and thus safeguard genome integrity. Among these is the piRNA pathway, a gonad-specific system based on small non-coding RNAs that recognise active transposons and instruct their silencing. Ultimately, piRNAs trigger epigenetic silencing of transposon loci. The work in this thesis investigates the molecular mechanisms by which piRNAs are produced from the correct substrates and how piRNAs can induce silencing of target loci in Drosophila melanogaster. First, I investigated how piRNA precursors are selected for processing and how they are transported to mitochondria, where piRNA production occurs. I explored the role of an uncharacterised Drosophila gene previously implicated in germline transposon control: CG10880/Daedalus (Daed). I found that Daed is an essential component of the mitochondrial piRNA biogenesis machinery and that it recruits the RNA helicase Armitage (Armi) to mitochondria. If Armi fails to be recruited, piRNA biogenesis cannot occur since Armi’s role is that of delivering piRNA precursors to the mitochondrial processing machinery. Secondly, I investigated how the major piRNA precursor in somatic cells, namely flamenco (flam) transcript, is exported and specified for downstream piRNA production. I uncovered that flam export is closely linked to the assembly of peri-nuclear condensates of the helicase fs(1)Yb (Yb). Furthermore, some subunits of the Nuclear Pore Complex (NPC) are also required for the production flam-derived piRNAs, thus suggesting the evolution of a specialised machinery that couples nuclear export and processing of this transcript. Finally, I set out to understand how piRNAs trigger silencing of active transposons. I found that Panoramix (Panx), the central effector of piRNA-guided epigenetic silencing, assembles into a complex with two other proteins: Nxf2 and Nxt1. We characterised the dependencies within the complex and found that all three components are essential to initiate silencing. Intriguingly, Nxf2 and Nxt1 belong to the family of nuclear export factors, thus suggesting that the piRNA pathway has co-opted proteins involved in RNA export and repurposed them for transposon control. Overall, this work provides new insights on the molecular mechanisms of piRNA-guided transposon silencing in Drosophila and shows evidence that transposon control pathways can exploit cellular factors for novel functions.

Remodeling of the gene regulatory network in cells is believed to be a prerequisite for their lineage reprogramming. However, its key regulatory factors are not yet elucidated. In this article, we investigate the role of PIWI proteins and provide evidence that one of them, MIWI2, is elicited during transdifferentiation of fibroblasts into hepatocyte‐like cells. In coincidence with the peak expression of MIWI2, we identified the appearance of a unique intermediate epigenetic state characterized by a specific Piwi‐interacting RNA (piRNA) profile consisting of 219 novel sequences. Knockout of MIWI2 greatly improved the formation of the induced hepatocytes, whereas overexpression of exogenous MIWI2 completely abolished the stimulated effect. A bioinformatics analysis of piRNA interaction network, followed by experimental validation, revealed the Notch signaling pathway as one of the immediate effectors of MIWI2. Altogether, our results show for the first time that temporal expression of MIWI2 contributes negatively to cell plasticity not only in germline, but also in developed cells, such as mouse fibroblasts. stem cells 2019;37:803–812, The role of MIWI2 in transdifferentiation of somatic cells unveils an intermediate cellular state characterized by the biogenesis of piRNAs. Jag1‐Notch signaling is shown to be one of the immediate downstream effector pathways. (iHep: induced hepatocyte. TFs: transcription factors)

Transposable elements (TEs) can damage genomes, thus organisms employ a variety of mechanisms to repress TE expression. However, these mechanisms often fail over time leading to de-repression of TEs in aging tissues. 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 epithelial stem cells ofHydra, a long-lived freshwater cnidarian.Hydrahave three stem cell populations; endodermal and ectodermal epithelial stem cells are strictly somatic, whereas the interstitial stem cells retain germline competence. In our previous study, we found that the PIWI proteins are expressed in all threeHydrastem cell types. In this study, we focus on the ectodermal and endodermal epithelial stem cells to understand the somatic function of the pathway. We isolated piRNAs fromHydrathat 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 inHydraepithelial stem cells. First, epithelial knockdown of theHydraPIWI proteinhywiresulted in upregulation 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. Interestingly, we found that RNAi knockdown ofhywileads to an upregulation of genes involved in innate immunity, which may be in response to TE upregulation; this is consistent with recent studies on TE expression in mammalian cells. Altogether, this study suggests a function for the PIWI-piRNA pathway in maintaining the long-lived somatic cell lineages ofHydraand may point to a broader role for this pathway in protecting somatic tissue from TE-induced damage.

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.

Background Aberrant overexpression of PIWI/piRNA pathway proteins is shown for many types of tumors. Interestingly, these proteins are downregulated in testicular germ cell tumors (TGCTs) compared to normal testis tissues. Here, we used germline and TGCT markers to assess the piRNA biogenesis and function in TGCTs and their precursor germ cell neoplasia in situ (GCNIS). Methods We used small RNA deep sequencing, qRT-PCR, and mining public RNAseq/small RNA-seq datasets to examine PIWI/piRNA gene expression and piRNA biogenesis at four stages of TGCT development: (i) germ cells in healthy testis tissues, (ii) germ cells in testis tissues adjacent to TGCTs, (iii) GCNIS cells and (iv) TGCT cells. To this end, we studied three types of samples: (a) healthy testis, (b) testis tissues adjacent to two types of TGCTs (seminomas and nonseminomas) and containing both germ cells and GCNIS cells, as well as (c) matching TGCT samples. Results Based on our analyses of small RNA-seq data as well as the presence/absence of expression correlation between PIWI/piRNA pathway genes and germline or TGCT markers, we can suggest that piRNA biogenesis is intact in germ cells present in healthy adult testes, and adjacent to TGCTs. Conversely, GCNIS and TGCT cells were found to lack PIWI/piRNA pathway gene expression and germline-like piRNA biogenesis. However, using an in vitro cell line model, we revealed a possible role for a short PIWIL2/HILI isoform expressed in TGCTs in posttranscriptional regulation of the youngest members of LINE and SINE classes of transposable elements. Importantly, this regulation is also implemented without involvement of germline-like biogenesis of piRNAs. Conclusions Though further studies are warranted, these findings suggest that the conventional germline-like PIWI/piRNA pathway is lost in transition from germ cells to GCNIS cells. Electronic supplementary material The online version of this article (10.1186/s12885-017-3945-6) contains supplementary material, which is available to authorized users.

In about half the cases of involuntary childlessness, a male infertility factor is involved. The PIWI-LIKE genes, a subclade of the Argonaute protein family, are involved in RNA silencing and transposon control in the germline. Knockout of murine Piwi-like 1 and 2 homologs results in complete infertility in males. The aim of this study was to analyze whether the mRNA expression of human PIWI-LIKE 1-4 genes is altered in ejaculated spermatozoa of men with impaired sperm characteristics. Ninety male participants were included in the study, among which 47 were with normozoospermia, 36 with impaired semen characteristics according to the World Health Organization (WHO) manual, 5th edition, and 7 with azoospermia serving as negative control for the PIWI-LIKE 1-4 mRNA expression in somatic cells in the ejaculate. PIWI-LIKE 1-4 mRNA expression in the ejaculated spermatozoa of the participants was measured by quantitative real-time PCR. In nonazoospermic men, PIWI-LIKE 1-4 mRNA was measurable in ejaculated spermatozoa in different proportions. PIWI-LIKE 1 (100.0%) and PIWI-LIKE 2 (49.4%) were more frequently expressed than PIWI-LIKE 3 (9.6%) and PIWI-LIKE 4 (15.7%). Furthermore, a decreased PIWI-LIKE 2 mRNA expression showed a significant correlation with a decreased sperm count (P = 0.022) and an increased PIWI-LIKE 1 mRNA expression with a decreased progressive motility (P = 0.048). PIWI-LIKE 1 and PIWI-LIKE 2 mRNA expression exhibited a significant association with impaired sperm characteristics and may be a useful candidate for the evaluation of the impact of PIWI-LIKE 1-4 mRNA expression on male infertility.

SUMMARY PIWI proteins are known as mediators of transposon silencing in animal germlines but are also found in adult pluripotent stem cells of highly regenerative animals, where they are essential for regeneration. Study of the nuclear PIWI protein SMEDWI-2 in the planarian somatic stem cell system reveals an intricate interplay between transposons and cell differentiation in which a subset of transposons is inevitably activated during cell differentiation, and the PIWI protein is required to regain control. Absence of SMEDWI-2 leads to tissue-specific transposon derepression related to cell-type-specific chromatin remodeling events and in addition causes reduced accessibility of lineage-specific genes and defective cell differentiation, resulting in fatal tissue dysfunction. Finally, we show that additional PIWI proteins provide a stem-cell-specific second layer of protection in planarian neoblasts. These findings reveal a far-reaching role of PIWI proteins and PIWI-interacting RNAs (piRNAs) in stem cell biology and cell differentiation., Graphical Abstract, In brief Chromatin reorganization is an essential part of cell differentiation but risks reactivation of silenced repetitive elements. Li et al. show that planarians use continuous surveillance by a nuclear PIWI protein to chaperone the chromatin transition from pluripotent to differentiated cell, which is crucial for the planarian’s impressive regeneration and homeostasis.

The MALAT1 (Metastasis-Associated Lung Adenocarcinoma Transcript 1) gene encodes a noncoding RNA that is processed into a long nuclear retained transcript (MALAT1) and a small cytoplasmic tRNA-like transcript (mascRNA). Using an RNA sequence- and structure-based covariance model, we identified more than 130 genomic loci in vertebrate genomes containing the MALAT1 3' end triple-helix structure and its immediate downstream tRNA-like structure, including 44 in the green lizard Anolis carolinensis. Structural and computational analyses revealed a co-occurrence of components of the 3' end module. MALAT1-like genes in Anolis carolinensis are highly expressed in adult testis, thus we named them testis-abundant long noncoding RNAs (tancRNAs). MALAT1-like loci also produce multiple small RNA species, including PIWI-interacting RNAs (piRNAs), from the antisense strand. The 3' ends of tancRNAs serve as potential targets for the PIWI-piRNA complex. Thus, we have identified an evolutionarily conserved class of long noncoding RNAs (lncRNAs) with similar structural constraints, post-transcriptional processing, and subcellular localization and a distinct function in spermatocytes.

Drosophila key nuclear piRNA silencing pathway protein Piwi of the Argonaute family has been classically studied as a factor controlling transposable elements and fertility. Piwi has been shown to concentrate in the nucleolus for reasons largely unknown. Ribosomal RNA is the main component of the nucleolus. In this work the effect of a piwi mutation on rRNA is described. This work led to three important conclusions: A mutation in piwi induces antisense 5S rRNA expression, a processing defect of 2S rRNA orthologous to the 3&prime, end of eukaryotic 5.8S rRNA, and accumulation of fragments of all five rRNAs in Drosophila melanogaster ovaries. Hypotheses to explain these phenomena are proposed, possibly involving the interaction of the components of the piRNA pathway with the RNA surveillance machinery.

RNA silencing refers to gene silencing pathways mediated by small non-coding RNAs, including microRNAs. Piwi-interacting RNAs (piRNAs) constitute the largest class of small non-coding RNAs in animal gonads, which repress transposons to protect the germline genome from the selfish invasion of transposons. Deterioration of the system causes DNA damage, leading to severe defects in gametogenesis and infertility. Studies using Drosophila ovaries show that piRNAs originate from specific genomic loci, termed piRNA clusters, and that in piRNA biogenesis, cluster transcripts are processed into mature piRNAs via three distinct pathways: initiator or responder for ping-pong piRNAs and trailing for phased piRNAs. piRNAs then assemble with PIWI members of the Argonaute family of proteins to form piRNA-induced RNA silencing complexes (piRISCs), the core engine of the piRNA-mediated silencing pathway. Upon piRISC assembly, the PIWI member, Piwi, is translocated to the nucleus and represses transposons co-transcriptionally by inducing local heterochromatin formation at target transposon loci.

PIWI-interacting RNAs (piRNAs) are small non-coding RNAs that repress transposons in animal germlines. This protects the genome from the invasive DNA elements. piRNA pathway failures lead to DNA damage, gonadal development defects, and infertility. Thus, the piRNA pathway is indispensable for the continuation of animal life. piRNA-mediated transposon silencing occurs in both the nucleus and cytoplasm while piRNA biogenesis is a solely cytoplasmic event. piRNA production requires a number of proteins, the majority of which localize to non-membranous organelles that specifically appear in the gonads. Other piRNA factors are localized on outer mitochondrial membranes. In situ RNA hybridization experiments show that piRNA precursors are compartmentalized into other non-membranous organelles. In this review, we summarize recent findings about the function of these organelles in the Drosophila piRNA pathway by focusing on their assembly and function.

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 loss of germ cells and mutants develop as sterile males, a phenotype that is shared with other PIWI pathway mutants. Tdrd9 is required for proper piRNA production and similar to Tdrd1 mutants, mainly small RNAs from RNA transposons are affected. Additionally we observe a nuage phenotype which suggests that Tdrd9 plays a role in the dynamics between different RNA granules. Although we identify many nuage proteins including the two zebrafish PIWI proteins, Zili and Ziwi, as possible interaction partners of Vasa, we don’t see a dramatic effect on piRNA production. This effect might be obscured though by the presence of maternally provided Vasa protein and RNA as maternally provided protein can still be detected in mutant germ cells at 3 weeks post fertilization. Gtsf1, another conserved factor involved in small RNA biology is subject of chapter 4 where we find that both gtsf homologs in zebrafish localize to perinuclear nuage and interact with PIWI proteins. Nevertheless we do not find an effect on piRNA production in gtsf1 mutants, but, similar to the situation in mouse, gtsf1 is required for spermiogenesis and male fertility.

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 loss of germ cells and mutants develop as sterile males, a phenotype that is shared with other PIWI pathway mutants. Tdrd9 is required for proper piRNA production and similar to Tdrd1 mutants, mainly small RNAs from RNA transposons are affected. Additionally we observe a nuage phenotype which suggests that Tdrd9 plays a role in the dynamics between different RNA granules. Although we identify many nuage proteins including the two zebrafish PIWI proteins, Zili and Ziwi, as possible interaction partners of Vasa, we don’t see a dramatic effect on piRNA production. This effect might be obscured though by the presence of maternally provided Vasa protein and RNA as maternally provided protein can still be detected in mutant germ cells at 3 weeks post fertilization. Gtsf1, another conserved factor involved in small RNA biology is subject of chapter 4 where we find that both gtsf homologs in zebrafish localize to perinuclear nuage and interact with PIWI proteins. Nevertheless we do not find an effect on piRNA production in gtsf1 mutants, but, similar to the situation in mouse, gtsf1 is required for spermiogenesis and male fertility.

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 loss of germ cells and mutants develop as sterile males, a phenotype that is shared with other PIWI pathway mutants. Tdrd9 is required for proper piRNA production and similar to Tdrd1 mutants, mainly small RNAs from RNA transposons are affected. Additionally we observe a nuage phenotype which suggests that Tdrd9 plays a role in the dynamics between different RNA granules. Although we identify many nuage proteins including the two zebrafish PIWI proteins, Zili and Ziwi, as possible interaction partners of Vasa, we don’t see a dramatic effect on piRNA production. This effect might be obscured though by the presence of maternally provided Vasa protein and RNA as maternally provided protein can still be detected in mutant germ cells at 3 weeks post fertilization. Gtsf1, another conserved factor involved in small RNA biology is subject of chapter 4 where we find that both gtsf homologs in zebrafish localize to perinuclear nuage and interact with PIWI proteins. Nevertheless we do not find an effect on piRNA production in gtsf1 mutants, but, similar to the situation in mouse, gtsf1 is required for spermiogenesis and male fertility.

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 loss of germ cells and mutants develop as sterile males, a phenotype that is shared with other PIWI pathway mutants. Tdrd9 is required for proper piRNA production and similar to Tdrd1 mutants, mainly small RNAs from RNA transposons are affected. Additionally we observe a nuage phenotype which suggests that Tdrd9 plays a role in the dynamics between different RNA granules. Although we identify many nuage proteins including the two zebrafish PIWI proteins, Zili and Ziwi, as possible interaction partners of Vasa, we don’t see a dramatic effect on piRNA production. This effect might be obscured though by the presence of maternally provided Vasa protein and RNA as maternally provided protein can still be detected in mutant germ cells at 3 weeks post fertilization. Gtsf1, another conserved factor involved in small RNA biology is subject of chapter 4 where we find that both gtsf homologs in zebrafish localize to perinuclear nuage and interact with PIWI proteins. Nevertheless we do not find an effect on piRNA production in gtsf1 mutants, but, similar to the situation in mouse, gtsf1 is required for spermiogenesis and male fertility.

More than a decade ago, PIWI-interacting RNA (piRNA) was discovered almost simultaneously by four different research groups. The length of this type of single stranded noncoding RNA is 24~31 nucleotides (nt), with most of the piRNAs fall into the range of 29~30 nt. PiRNAs form specific RNA-induced silencing complex with PIWI subfamily proteins, which is how piRNA got its name. PiRNAs are initially famous for the crucial roles they played in germline cells. Binding with PIWI family proteins, piRNAs is able to affect methylation of genomic DNA in germline cells and, therefore, maintaining genomic stability and suppressing transposons. Since mammalian PIWI subfamily proteins are mainly germline specific, it was once thought that piRNAs might only function in the gonadal cells. However, evidence from the later research suggest that piRNAs are broadly expressed in many kinds of somatic cells and are involved in numerous pathological condition far beyond those have been reported in the germline. For example, piRNAs were discovered to be abnormally expressed in several kinds of cancers. PiRNAs are also shown to be promising prognostic markers for various types of cancers. Interestingly, a recent research indicated that piRNAs are also regulators for pancreatic beta cell function. PiRNAs are promising regulators for the development of type 2 diabetes. From a disease-oriented point of view, this review will focus on both confirmed and proposed biological functions of piRNAs mostly in those fields beyond germline cells. Meanwhile, some of the underlying molecular mechanisms will also be mentioned.

Piwi proteins are important for germ cell development in most animals. These proteins are guided to specific targets by small guide RNAs, referred to as piRNAs or 21U RNAs in Caenorhabditis elegans. In this organism, even though genetic screens have uncovered 21U RNA biogenesis factors, little is known about how these factors interact or what they do. Based on the previously identified 21U biogenesis factor PID-1 (piRNA-induced silencing-defective 1), we here define a novel protein complex, PETISCO (PID-3, ERH-2, TOFU-6, and IFE-3 small RNA complex), that is required for 21U RNA biogenesis. PETISCO contains both potential 5′ cap and 5′ phosphate RNA-binding domains and interacts with capped 21U precursor RNA. We resolved the architecture of PETISCO and revealed a second function for PETISCO in embryonic development. This essential function of PETISCO is mediated not by PID-1 but by the novel protein TOST-1 (twenty-one U pathway antagonist). In contrast, TOST-1 is not essential for 21U RNA biogenesis. Both PID-1 and TOST-1 interact directly with ERH-2 using a conserved sequence motif. Finally, our data suggest a role for TOST-1:PETISCO in SL1 homeostasis in the early embryo. Our work describes a key complex for 21U RNA processing in C. elegans and strengthens the view that 21U RNA biogenesis is built on an snRNA-related pathway.

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 of different viruses. NIH [AI067380] The authors would like to thank Dr. Aaron Brault, Centers for Disease Control and prevention, for providing Hsu cells, CT cells, and the Calbertado virus sequence. The mosquito used in the graphical abstract was provided by Ana L. Ramirez through figshare (Ramirez, 2019). This study was funded by NIH grant AI067380.

Macromolecular complexes and organelles play crucial roles within cells, but their native architectures are often unknown. Here, we use an evolutionarily conserved germline organelle, the germ granule, as a paradigm. In Drosophila embryos, we map one of its interactomes using a novel in vivo crosslinking approach that employs two interacting granule proteins and determines their common neighbor molecules. We identified an in vivo granule assembly of Tudor, Aubergine, motor and metabolic proteins, and RNA helicases, and provide evidence for direct interactions within this assembly using purified components. Our study indicates that germ granules contain efficient biochemical reactors involved in post-transcriptional gene regulation.

Piwi is necessary for germ cell development in a diverse range of organisms and homologues have been commonly used to identify primordial germ cells (PGCs) during embryogenesis. Here, we isolated full-length cDNA of Piwi ortholog in the Pacific oyster Crassostrea gigas and characterized its expression patterns (Cg-Piwi-like) along with analyzing the expression alternation in the gonad of diploids compared with those sterile triploids. qPCR showed that the transcript of Cg-Piwi-like was mainly restricted to the gonad in diploids with ovarian tissues of triploids showing the highest expression. in situ hybridization revealed that Cg-Piwi-like was found in both female and male gonad where the strongest expression was shown in the germ cells at early stages with no signal in somatic cells. Whole-mount in situ hybridization suggested Cg-Piwi-like was maternally deposited and the localization of Cg-Piwi-like mRNA in mesodermal cells might be the putative PGCs in C. gigas. These results suggest that Cg-Piwi-like was involved in germ line formation, differentiation, and maintenance of germ cells in C. gigas. The obtained findings provide valuable evidences to further facilitate identification of the putative PGCs in the Pacific oyster using Cg-Piwi-like as a molecular marker.

PIWI proteins and their guiding Piwi-interacting (pi-) RNAs direct the silencing of target nucleic acids in the animal germline and soma. Although in mammal testes fetal piRNAs are involved in extensive silencing of transposons, pachytene piRNAs have additionally been shown to act in post-transcriptional gene regulation. The bulk of pachytene piRNAs is produced from large genomic loci, named piRNA clusters. Recently, the presence of reversed pseudogenes within piRNA clusters prompted the idea that piRNAs derived from such sequences might direct regulation of their parent genes. Here, we examine primate piRNA clusters and integrated pseudogenes in a comparative approach to gain a deeper understanding about mammalian piRNA cluster evolution and the presumed gene-regulatory role of pseudogene-derived piRNAs. Initially, we provide a broad analysis of the evolutionary relationships of piRNA clusters and their differential activity among six primate species. Subsequently, we show that pseudogenes in reserve orientation relative to piRNA cluster transcription direction generally do not exhibit signs of selection pressure and cause weakly conserved targeting of homologous genes among species, suggesting a lack of functional constraints and thus only a minor significance for gene regulation in most cases. Finally, we report that piRNA-producing loci generally tend to be located in active genomic regions with elevated gene and pseudogene density. Thus, we conclude that the presence of most pseudogenes in piRNA clusters might be regarded as a byproduct of piRNA cluster generation, whereas this does not exclude that some pseudogenes nevertheless play critical roles in individual cases.