Distinct sets of PIWI proteins produce arbovirus and transposon-derived piRNAs in Aedes aegypti mosquito cells

The PIWI-interacting RNA (piRNA) pathway is essential for transposon silencing in many model organisms. Its remarkable efficiency relies on a sophisticated amplification mechanism known as the ping-pong loop. In Alphavirus-infected Aedes mosquitoes, piRNAs with sequence features that suggest ping-pong-dependent biogenesis are produced from viral RNA. The PIWI family in Aedes mosquitoes is expanded when compared to other model organisms, raising the possibility that individual PIWI proteins have functionally diversified in these insects. Here, we show that Piwi5 and Ago3, but none of the other PIWI family members, are essential for piRNA biogenesis from Sindbis virus RNA in infected Aedes aegypti cells. In contrast, the production of piRNAs from transposons relies on a more versatile set of PIWI proteins, some of which do not contribute to viral piRNA biogenesis. These results indicate that functional specialization allows distinct mosquito PIWI proteins to process RNA from different endogenous and exogenous sources.     

Mouse primordial germ-cell-like cells lack piRNAs

1. Download : Download high-res image (159KB)
2. Download : Download full-size image

     

Endogenous siRNAs and piRNAs derived from transposable elements and genes in the malaria vector mosquito Anopheles gambiae

Background

The siRNA and piRNA pathways have been shown in insects to be essential for regulation of gene expression and defence against exogenous and endogenous genetic elements (viruses and transposable elements). The vast majority of endogenous small RNAs produced by the siRNA and piRNA pathways originate from repetitive or transposable elements (TE). In D. melanogaster, TE-derived endogenous siRNAs and piRNAs are involved in genome surveillance and maintenance of genome integrity. In the medically relevant malaria mosquito Anopheles gambiae TEs constitute 12-16% of the genome size. Genetic variations induced by TE activities are known to shape the genome landscape and to alter the fitness in An. gambiae.

Results

Here, using bioinformatics approaches we analyzed the small RNA data sets from 6 libraries formally reported in a previous study and examined the expression of the mixed germline/somatic siRNAs and piRNAs produced in adult An. gambiae females. We characterized a large population of TE-derived endogenous siRNAs and piRNAs, which constitutes 56-60% of the total siRNA and piRNA reads in the analysed libraries. Moreover, we identified a number of protein coding genes producing gene-specific siRNAs and piRNAs that were generally expressed at much lower levels than the TE-associated small RNAs. Detailed sequence analysis revealed that An. gambiae piRNAs were produced by both “ping-pong” dependent (TE-associated piRNAs) and independent mechanisms (genic piRNAs). Similarly to D. melanogaster, more than 90% of the detected piRNAs were produced from TE-associated clusters in An. gambiae. We also found that biotic stress as blood feeding and infection with Plasmodium parasite, the etiological agent of malaria, modulated the expression levels of the endogenous siRNAs and piRNAs in An. gambiae.

Conclusions

We identified a large and diverse set of the endogenously derived siRNAs and piRNAs that share common and distinct aspects of small RNA expression across insect species, and inferred their impact on TE and gene activity in An. gambiae. The TE-specific small RNAs produced by both the siRNA and piRNA pathways represent an important aspect of genome stability and genetic variation, which might have a strong impact on the evolution of the genome and vector competence in the malaria mosquitoes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1436-1) contains supplementary material, which is available to authorized users.     

Expression of a heat-inducible gene in transfected mosquito cells

The evolutionary conservation of the heat shock response suggests that plasmids containing promoters from Drosophila heat shock protein (hsp) genes will be useful in the development of gene transfer procedures for cell lines representing a variety of insect species. Conditions for induction of endogenous hsp genes and for expression of the chloramphenicol acetyltransferase (CAT) gene regulated by the Drosophila hsp 70 promoter were examined in Aedes albopictus (mosquito) cells. Five hsps, ranging in size from 27,000 to 90,000 D, were induced in A. albopictus cells during incubation at 41°C in medium containing [³⁵S]methionine. Relative synthesis of these proteins at 37 and 41°C indicated that Aedes hsp 66 is homologous to Drosophila hsp 70. Detection of CAT activity in transfected mosquito cells was enhanced 10-fold under heat shock conditions (6 h, 41°C) based on maximal expression of hsp 66, relative to conditions defined for expression of hsp 70 in Drosophila cells. Analysis of the endogenous heat shock response may be essential to the optimal use of plasmids containing the Drosophila hsp 70 promoter with other insect cell types.     

Maternally deposited germline piRNAs silence the tirant retrotransposon in somatic cells

Transposable elements (TEs), whose propagation can result in severe damage to the host genome, are silenced in the animal gonad by Piwi‐interacting RNAs (piRNAs). piRNAs produced in the ovaries are deposited in the embryonic germline and initiate TE repression in the germline progeny. Whether the maternally transmitted piRNAs play a role in the silencing of somatic TEs is however unknown. Here we show that maternally transmitted piRNAs from the tirant retrotransposon in Drosophila are required for the somatic silencing of the TE and correlate with an increase in histone H3K9 trimethylation an active tirant copy.     

Determination of half-reaction equilibrium in a ping-pong enzyme mechanism

Substituted enzyme (or ping-pong) mechanisms usually involve enzymes that exist in two forms that alternate during the catalytic reaction. A method is described here for determining the position of the equilibrium of a half reaction in a ping-pong enzyme mechanism that is based on the kinetics of the burst reaction which occurs upon addition of reactants that recycle the enzyme from one form to another. The theoretical basis for the analysis is developed, and the method is applied to the half reaction of the aldimine form of aspartate transaminase with difluoro-oxaloacetate. Special issue dedicated to Herman Bachelard     

An osmoregulatory syncytium and associated cells in a freshwater mosquito

In embryological terms the anal papillae are the product of eversion of the hindgut tissues. The rectum and the anal papillae have the same origin and have a marked structural similarity. The insect hindgut is very labile being able to produce salt transporting or ‘chloride cells’ from any of the tissues of which it is composed.The hindgut consists of four distinct regions: the ileum and part of the anal canal have a mechanical function, the rectum and the posterior anal canal contain transporting cells. Two new cell types, ‘interstitial’ and ‘tertiary’ are reported in the rectum.The structure of the anal papillae changes with increased salinity. Changes in the plasma membranes alter the surface area for transport. Changes in the number of mitochondria are not accompanied by changes in oxygen consumption. If mitochondria are the site of oxidative metabolism then their number docs not control the level of oxygen consumption.In Aedes aegypti the papillary epithelium appears to be a syncytium. Across the lumen of the papillae there are cellular sheets supporting the tracheoles. At the base of the papillae there is a cellular transition zone; circular muscles in this region may be used to occlude the papillae. The control of salt transport may be hormonal.     

Profiling Sex-Specific piRNAs in Zebrafish

Piwi proteins and their partner small RNAs play an essential role in fertility, germ-line stem cell development, and the basic control and evolution of animal genomes. However, little knowledge exists regarding piRNA biogenesis. Utilizing microfluidic chip analysis, we present a quantitative profile of zebrafish piRNAs expressed differentially between testis and ovary. The sex-specific piRNAs are derived from separate loci of repeat elements in the genome. Ovarian piRNAs can be categorized into groups that reach up to 92 members, indicating a sex-specific arrangement of piRNA genes in the genome. Furthermore, precursor piRNAs preferentially form a hairpin structure at the 3′end, which seem to favor the generation of mature sex-specific piRNAs. In addition, the mature piRNAs from both the testis and the ovary are 2′-O-methylated at their 3′ ends.SMALL RNAs, ranging from 19 to 30 nucleotides (nt) in length, constitute a large family of regulatory molecules with diverse functions in invertebrates, vertebrates, plants, and fungi (Bartel 2004; Nakayashiki 2005). Two major classes of small RNAs are microRNAs (miRNAs) and small interfering RNAs (siRNAs). The functions of small RNAs have been conserved through evolution; they have been shown to inhibit gene expression at the levels of mRNA degradation, translational repression, chromatin modification, heterochromatin formation, and DNA elimination (Mochizuki et al. 2002; Bartel 2004; Kim et al. 2005; Brodersen and Voinnet 2006; Lee and Collins 2006; Vaucheret 2006).Over the past few years, focus on the genetics of small RNAs has helped clarify the mechanisms behind the regulation of these molecules. While hundreds of small RNAs have been identified from mammalian somatic tissues, relatively little is known about small RNAs in germ cells. A recent breakthrough has been the identification of small RNAs that associate with Piwi proteins (piRNAs) from Drosophila and mammalian gonads (Aravin et al. 2001, 2006; Girard et al. 2006; Grivna et al. 2006; Vagin et al. 2006; Watanabe et al. 2006). piRNAs and their interacting proteins Ziwi/Zili have also been identified in zebrafish (Houwing et al. 2007, 2008). Increasing evidence indicates that piRNAs play roles mainly in germ cell differentiation and genomic stability (Carthew 2006; Lau et al. 2006; Vagin et al. 2006; Brennecke et al. 2007; Chambeyron et al. 2008; Klattenhoff and Theurkauf 2008; Kuramochi-Miyagawa et al. 2008; Kim et al. 2009; Lim et al. 2009; Unhavaithaya et al. 2009). Moreover, although piRNAs are mostly expressed in germ line cells, recent studies showed piRNA expression in nongerm cells, for example, T-cell lines (Jurkat cells and MT4) (Azuma-Mukai et al. 2008; Yeung et al. 2009), indicating other functions such as in the immune system. piRNAs do not appear to be derived from double-stranded RNA precursors, and their biogenesis mechanisms, although unclear, may be distinct from those of siRNA and miRNA. Recently, two distinct piRNA production pathways were further proposed: the “ping-pong” model (Brennecke et al. 2007; Gunawardane et al. 2007) and the Ago3-independent piRNA pathway centered on Piwi in somatic cells (Li et al. 2009; Malone et al. 2009). However, the mechanistic pathways of piRNA activity and their biogenesis are still largely unknown.Teleost fishes comprise >24,000 species, accounting for more than half of extant vertebrate species, displaying remarkable variation in morphological and physiological adaptations (see review in Zhou et al. 2001). Recently, Houwing et al. (2007, 2008) reported findings on Ziwi/Zili and associated piRNAs, implicating roles in germ cell differentiation, meiosis, and transposon silencing in the germline of the zebrafish. However, some of the identified zebrafish piRNAs are nonrepetitive and nontransposon-related piRNAs, suggesting that piRNAs may have additional unknown roles. In this study, we show that for males and females, piRNAs are specifically derived from separate loci of the repeat elements, and that ovarian piRNAs are far more often associated in groups. Genomic analysis of piRNAs indicates a tendency to folding at the 3′ end of the piRNA precursor, which may favor cleavage of the piRNA precursor to generate mature sex-specific piRNAs. Furthermore, methylation modification occurs at the 2′-O-hydroxyl group on the ribose of the final 3′ nucleotide in both the testis and the ovary.     

The human Piwi protein Hiwi2 associates with tRNA-derived piRNAs in somatic cells

The Piwi-piRNA pathway is active in animal germ cells where its functions are required for germ cell maintenance and gamete differentiation. Piwi proteins and piRNAs have been detected outside germline tissue in multiple phyla, but activity of the pathway in mammalian somatic cells has been little explored. In particular, Piwi expression has been observed in cancer cells, but nothing is known about the piRNA partners or the function of the system in these cells. We have surveyed the expression of the three human Piwi genes, Hiwi, Hili and Hiwi2, in multiple normal tissues and cancer cell lines. We find that Hiwi2 is ubiquitously expressed; in cancer cells the protein is largely restricted to the cytoplasm and is associated with translating ribosomes. Immunoprecipitation of Hiwi2 from MDAMB231 cancer cells enriches for piRNAs that are predominantly derived from processed tRNAs and expressed genes, species which can also be found in adult human testis. Our studies indicate that a Piwi-piRNA pathway is present in human somatic cells, with an uncharacterised function linked to translation. Taking this evidence together with evidence from primitive organisms, we propose that this somatic function of the pathway predates the germline functions of the pathway in modern animals.     

Teratocarcinoma cells exhibit growth cooperativity in vitro

Malignant PC13 embryonal carcinoma (EC) cells differentiate in vitro in response to retinoic acid, giving rise to a population of benign endoderm-like cells (END), termed PC13 END. PC13 EC and PC13 END cells exhibit growth cooperativity in co-culture, whereby the EC cells stimulate END cell proliferation and the END cells can support EC cell multiplication. The EC cells' stimulatory effect operates via soluble, diffusible factors which are also active on a range of fibroblast cell lines. END cells support the multiplication of EC cells plated at low density, via a multifactorial mechanism. Contact-dependent effects can operate in the absence of END cell metabolic activity, while contact-independent effects require the continuous presence of live END cells. It was observed that there was a variation in the ability of fibroblast cell lines to act as EC cell feeders. Similar interactive events may be important during the in vivo proliferation and differentiation of teratocarcinoma cells and their embryonic counterparts.     

Persistent,triple-virus co-infections in mosquito cells

Background  

It is known that insects and crustaceans can carry simultaneous, active infections of two or more viruses without showing signs of disease, but it was not clear whether co-infecting viruses occupied the same cells or different cells in common target tissues. Our previous work showed that successive challenge of mosquito cell cultures followed by serial, split-passage resulted in stabilized cultures with 100% of the cells co-infected with Dengue virus (DEN) and an insect parvovirus (densovirus) (DNV). By addition of Japanese encephalitis virus (JE), we tested our hypothesis that stable, persistent, triple-virus co-infections could be obtained by the same process.     

Genetic changes in methotrexate-resistant mosquito cells.

A stepwise selection procedure was used to obtain from Mtx-5011 Aedes albopictus cells, variants with increased resistance to methotrexate (mtx). On the basis of growth, the Mtx-5011 derivatives were 270- to 3,000-fold more resistant to mtx than wild-type mosquito cells. Properties associated with mtx resistance in these cells were consistent with amplification of the dihydrofolate reductase (DHFR) gene. The cells overproduced DHFR protein, were enriched with DHFR mRNA, and DNA from resistant cells was enriched for a band that likely contained the DHFR coding sequence. Karyotype analysis indicated that high levels of resistance were accompanied by a conversion to tetraploidy, chromosome rearrangements, and an apparent duplication of one of the mosquito chromosomes.