Somatic piRNAs and Transposons are Differentially Expressed Coincident with Skeletal Muscle Atrophy and Programmed Cell Death

PIWI-interacting RNAs (piRNAs) are small single-stranded RNAs that can repress transposon expression via epigenetic silencing and transcript degradation. They have been identified predominantly in the ovary and testis, where they serve essential roles in transposon silencing in order to protect the integrity of the genome in the germline. The potential expression of piRNAs in somatic cells has been controversial. In the present study we demonstrate the expression of piRNAs derived from both genic and transposon RNAs in the intersegmental muscles (ISMs) from the tobacco hawkmoth Manduca sexta. These piRNAs are abundantly expressed, ∼27 nt long, map antisense to transposons, are oxidation resistant, exhibit a 5’ uridine bias, and amplify via the canonical ping-pong pathway. An RNA-seq analysis demonstrated that 19 piRNA pathway genes are expressed in the ISMs and are developmentally regulated. The abundance of piRNAs does not change when the muscles initiate developmentally-regulated atrophy, but are repressed coincident with the commitment of the muscles undergo programmed cell death at the end of metamorphosis. This change in piRNA expression is correlated with the repression of several retrotransposons and the induction of specific DNA transposons. The developmentally-regulated changes in the expression of piRNAs, piRNA pathway genes, and transposons are all regulated by 20-hydroxyecdysone, the steroid hormone that controls the timing of ISM death. Taken together, these data provide compelling evidence for the existence of piRNA in somatic tissues and suggest that they may play roles in developmental processes such as programmed cell death.

Transposable Element Expression and Regulation Profile in Gonads of Interspecific Hybrids of Drosophila arizonae and Drosophila mojavensis wrigleyi

Interspecific hybridization may lead to sterility and/or inviability through differential expression of genes and transposable elements (TEs). In Drosophila, studies have reported massive TE mobilization in hybrids from interspecific crosses of species presenting high divergence times. However, few studies have examined the consequences of TE mobilization upon hybridization in recently diverged species, such as Drosophila arizonae and D. mojavensis. We have sequenced transcriptomes of D. arizonae and the subspecies D. m. wrigleyi and their reciprocal hybrids, as well as piRNAs, to analyze the impact of genomic stress on TE regulation. Our results revealed that the differential expression in both gonadal tissues of parental species was similar. Globally, ovaries and testes showed few deregulated TEs compared with both parental lines. Analyses of small RNA data showed that in ovaries, the TE upregulation is likely due to divergence of copies inherited from parental genomes and lack of piRNAs mapping to them. Nevertheless, in testes, the divergent expression of genes associated with chromatin state and piRNA pathway potentially indicates that TE differential expression is related to the divergence of regulatory genes that play a role in modulating transcriptional and post-transcriptional mechanisms.

Complex Genetic Interactions between Piwi and HP1a in the Repression of Transposable Elements and Tissue-Specific Genes in the Ovarian Germline

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.

A transposon expression burst accompanies the activation of Y-chromosome fertility genes during Drosophila spermatogenesis

Transposable elements (TEs) must replicate in germline cells to pass novel insertions to offspring. In Drosophila melanogaster ovaries, TEs can exploit specific developmental windows of opportunity to evade host silencing and increase their copy numbers. However, TE activity and host silencing in the distinct cell types of Drosophila testis are not well understood. Here, we reanalyze publicly available single-cell RNA-seq datasets to quantify TE expression in the distinct cell types of the Drosophila testis. We develop a method for identification of TE and host gene expression modules and find that a distinct population of early spermatocytes expresses a large number of TEs at much higher levels than other germline and somatic components of the testes. This burst of TE expression coincides with the activation of Y chromosome fertility factors and spermatocyte-specific transcriptional regulators, as well as downregulation of many components of the piRNA pathway. The TEs expressed by this cell population are specifically enriched on the Y chromosome and depleted on the X chromosome, relative to other active TEs. These data suggest that some TEs may achieve high insertional activity in males by exploiting a window of opportunity for mobilization created by the activation of spermatocyte-specific and Y chromosome-specific transcriptional programs.

Aedes aegypti Piwi4 Structural Features Are Necessary for RNA Binding and Nuclear Localization

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.

Daphnia magna egg piRNA cluster expression profiles change as mothers age.

PiRNAs prevent transposable elements wreaking havoc on the germline genome. Changes in piRNA expression over the lifetime of an individual may impact on ageing through continued suppression, or release, of transposable element expression. We identified piRNA producing clusters in the genome of Daphnia magna by a combination of bioinformatic methods, and then contrasted their expression between parthenogenetically produced eggs representing maternally-deposited germline piRNAs of young (having their 1st clutch) and old (having their 5th clutch) mothers. Results from eggs were compared to cluster expression in three generations of adults. As for other arthropods, D. magna encodes long uni-directionally transcribed non-coding RNAs which consist of transposable element fragments which account for most piRNAs expressed. Egg tissues showed extensive differences between clutches from young mothers and those from old mothers, with 578 and 686 piRNA clusters upregulated, respectively, although most log fold-change differences for significant clusters were modest. When considering only highly expressed clusters, there was a bias towards 1st clutch eggs at 41 upregulated versus eight clusters in the eggs from older mothers. F0 generation differences between young and old mothers were fewer than eggs, as 179 clusters were up-regulated in young versus 170 old mothers. This dropped to 31 versus 22 piRNA clusters when comparing adults in the F1 generation, and no differences were detected in the F3 generation. These patterns were similar to that observed for D. magna micro-RNA expression. Little overlap in differentially expressed clusters was found between adults containing mixed somatic and germline (ovary) tissues and germ-line representing eggs. A cluster encompassing a Tudor domain containing gene important in the piRNA pathway was upregulated in the eggs from old mothers. We hypothesise that regulation of this gene this could form part of a feedback loop that reduces piRNA pathway activity explaining the reduced number of highly-expressed clusters in eggs from old mothers.

A genetic toolkit for studying transposon control in the Drosophila melanogaster ovary

Argonaute proteins of the PIWI clade complexed with PIWI-interacting RNAs (piRNAs) protect the animal germline genome by silencing transposable elements. One of the leading experimental systems for studying piRNA biology is the Drosophila melanogaster ovary. In addition to classical mutagenesis, transgenic RNA interference (RNAi), which enables tissue-specific silencing of gene expression, plays a central role in piRNA research. Here, we establish a versatile toolkit focused on piRNA biology that combines germline transgenic RNAi, GFP marker lines for key proteins of the piRNA pathway, and reporter transgenes to establish genetic hierarchies. We compare constitutive, pan-germline RNAi with an equally potent transgenic RNAi system that is activated only after germ cell cyst formation. Stage-specific RNAi allows us to investigate the role of genes essential for germline cell survival, for example nuclear RNA export or the SUMOylation pathway, in piRNA-dependent and independent transposon silencing. Our work forms the basis for an expandable genetic toolkit provided by the Vienna Drosophila Resource Center.

MORC3, a novel MIWI2 association partner, as an epigenetic regulator of piRNA dependent transposon silencing in male germ cells

The PIWI (P-element-induced wimpy testis)-interacting-RNA (piRNA) pathway plays a crucial role in the repression of TE (transposable element) expression via de novo DNA methylation in mouse embryonic male germ cells. Various proteins, including MIWI2 are involved in the process. TE silencing is ensured by piRNA-guided MIWI2 that recruits some effector proteins of the DNA methylation machinery to TE regions. However, the molecular mechanism underlying the methylation is complex and has not been fully elucidated. Here, we identified MORC3 as a novel associating partner of MIWI2 and also a nuclear effector of retrotransposon silencing via piRNA-dependent de novo DNA methylation in embryonic testis. Moreover, we show that MORC3 is important for transcription of piRNA precursors and subsequently affects piRNA production. Thus, we provide the first mechanistic insights into the role of this effector protein in the first stage of piRNA biogenesis in embryonic TE silencing mechanism.

To export, or not to export: how nuclear export factor variants resolve Piwi's dilemma

Piwi-interacting RNAs (piRNAs) defend animal gonads by guiding PIWI-clade Argonaute proteins to silence transposons. The nuclear Piwi/piRNA complexes confer transcriptional repression of transposons, which is accompanied with heterochromatin formation at target loci. On the other hand, piRNA clusters, genomic loci that transcribe piRNA precursors composed of transposon fragments, are often recognized by piRNAs to define their heterochromatic identity. Therefore, Piwi/piRNA complexes must resolve this conundrum of silencing transposons while allowing the expression of piRNA precursors, at least in Drosophila germlines. This review is focused on recent advances how the piRNA pathway deals with this genetic conflict.

Coupled protein synthesis and ribosome-guided piRNA processing on mRNAs

PIWI-interacting small RNAs (piRNAs) protect the germline genome and are essential for fertility. piRNAs originate from transposable element (TE) RNAs, long non-coding RNAs, or 3´ untranslated regions (3´UTRs) of protein-coding messenger genes, with the last being the least characterized of the three piRNA classes. Here, we demonstrate that the precursors of 3´UTR piRNAs are full-length mRNAs and that post-termination 80S ribosomes guide piRNA production on 3´UTRs in mice and chickens. At the pachytene stage, when other co-translational RNA surveillance pathways are sequestered, piRNA biogenesis degrades mRNAs right after pioneer rounds of translation and fine-tunes protein production from mRNAs. Although 3´UTR piRNA precursor mRNAs code for distinct proteins in mice and chickens, they all harbor embedded TEs and produce piRNAs that cleave TEs. Altogether, we discover a function of the piRNA pathway in fine-tuning protein production and reveal a conserved piRNA biogenesis mechanism that recognizes translating RNAs in amniotes.