Tandem repeat elements such as the highly diverse class of satellite repeats occupy large parts of eukaryotic chromosomes. Most occur at (peri)centromeric and (sub)telomeric regions and have been implicated in chromosome organization, stabilization, and segregation 1. Others are located more dispersed throughout the genome, but their functions remained largely unknown. Satellite repeats in euchromatic regions were hypothesized to regulate gene expression in cis by modulation of the local heterochromatin, or in trans via repeat-derived transcripts 2,3. Yet, due to a lack of

PIWI-interacting RNAs (piRNAs) comprise a class of small RNAs best known for suppressing transposable elements in germline tissues. The vector mosquito Aedes aegypti encodes seven PIWI genes, four of which are somatically expressed. This somatic piRNA pathway generates piRNAs from viral RNA during infection with cytoplasmic RNA viruses through ping-pong amplification by the PIWI proteins Ago3 and Piwi5. Yet, additional insights into the molecular mechanisms mediating non-canonical piRNA production are lacking. TUDOR-domain containing (Tudor) proteins facilitate piRNA biogenesis in Drosophila melanogaster and other model organisms. We thus hypothesized that Tudor proteins are required for viral piRNA production and performed a knockdown screen targeting all A. aegypti Tudor genes. Knockdown of the Tudor genes AAEL012437, Vreteno, Yb, SMN and AAEL008101-RB resulted in significantly reduced viral piRNA levels, with AAEL012437-depletion having the strongest effect. This protein, which we named Veneno, associates directly with Ago3 in an sDMA-dependent manner and localizes in cytoplasmic foci reminiscent of piRNA processing granules of Drosophila. Veneno-interactome analyses reveal a network of co-factors including the orthologs of the Drosophila piRNA pathway components Vasa and Yb, which in turn interacts with Piwi5. We propose that Veneno assembles a multi-protein complex for ping-pong dependent piRNA production from viral RNA.

The piRNA pathway is of key importance in controlling transposable elements in most animal species. In the vector mosquito Aedes aegypti, the presence of eight PIWI proteins and the accumulation of viral piRNAs upon arbovirus infection suggest additional functions of the piRNA pathway beyond genome defense. To better understand the regulatory potential of this pathway, we analyzed in detail host-derived piRNAs in A. aegypti Aag2 cells. We show that a large repertoire of protein-coding genes and non-retroviral integrated RNA virus elements are processed into genic piRNAs by different combinations of PIWI proteins. Among these, we identify a class of genes that produces piRNAs from coding sequences in an Ago3- and Piwi5-dependent fashion. We demonstrate that the replication-dependent histone gene family is a genic source of ping-pong dependent piRNAs and that histone-derived piRNAs are dynamically expressed throughout the cell cycle, suggesting a role for the piRNA pathway in the regulation of histone gene expression. Moreover, our results establish the Aag2 cell line as an accessible experimental model to study gene-derived piRNAs.

As in most arthropods, the PIWI-interacting RNA (piRNA) pathway in the vector mosquito Aedes aegypti is active in diverse biological processes in both soma and germline. To gain insights into piRNA biogenesis and effector complexes, we mapped the interactomes of the somatic PIWI proteins Ago3, Piwi4, Piwi5, and Piwi6 and identify numerous specific interactors as well as cofactors associated with multiple PIWI proteins. We describe the Piwi5 interactor AAEL014965, the direct ortholog of the Drosophila splicing factor pasilla. We find that Ae. aegypti Pasilla encodes a nuclear isoform and a cytoplasmic isoform, the latter of which is required for efficient piRNA production. In addition, we characterize a splice variant of the Tudor protein AAEL008101/Atari that associates with Ago3 and forms a scaffold for PIWI proteins and target RNAs to promote ping-pong amplification of piRNAs. Our study provides a useful resource for follow-up studies of somatic piRNA biogenesis, mechanism, and function in Aedes mosquitoes.

26Tandem repeat elements such as the highly diverse class of satellite repeats occupy large parts of 27 eukaryotic chromosomes. Most occur at (peri)centromeric and (sub)telomeric regions and have 28 been implicated in chromosome organization, stabilization, and segregation 1 . Others are located 29 more dispersed throughout the genome, but their functions remained largely enigmatic. Satellite 30 repeats in euchromatic regions were hypothesized to regulate gene expression in cis by modulation 31 of the local heterochromatin, or in trans via repeat-derived transcripts 2,3 . Yet, due to a lack of 32 experimental models, gene regulatory potential of satellite repeats remains largely unexplored. Here 33 we show that, in the vector mosquito Aedes aegypti, a satellite repeat promotes sequence-specific 34 gene silencing via the expression of two abundant PIWI-interacting RNAs (piRNAs). Strikingly, 35whereas satellite repeats and piRNA sequences generally evolve extremely fast 4-6 , this locus was 36 conserved for approximately 200 million years, suggesting a central function in mosquito biology. 37Tandem repeat-derived piRNA production commenced shortly after egg-laying and inactivation of 38 the most abundant of the two piRNAs in early embryos resulted in an arrest of embryonic 39 development. Transcriptional profiling in these embryos revealed the failure to degrade maternally 40 provided transcripts that are normally cleared during maternal-to-zygotic transition. Our results 41 reveal a novel mechanism in which satellite repeats regulate global gene expression in trans via 42 piRNA-mediated gene silencing, which is fundamental to embryonic development. These findings 43 highlight the regulatory potential of this enigmatic class of repeats. 44 45Main 46Even though satellite repeats have been discovered nearly 60 years ago 7,8 , and comprise a 47 substantial portion of eukaryotic genomes, little is known about the functions of this class of 48 repetitive DNA. Many satellite repeats are actively transcribed, and some of them produce small 49 interfering (si)RNAs required for the establishment and maintenance of heterochromatic regions 9-16 . 50