Chromatin profiling identifies transcriptional readthrough as a conserved mechanism for piRNA cluster biogenesis in mosquitoes

Abstract

The PIWI-interacting RNA (piRNA) pathway in culicine mosquitoes differs substantially from other model organisms in terms of an expanded PIWI gene family and ubiquitously expressed piRNAs in somatic tissues. Here, through comprehensive analyses of endogenous piRNAs in Aedes aegypti, we defined core piRNA clusters as small RNA source loci that showed ubiquitous expression in both somatic and germline tissues. These core piRNA clusters produced piRNAs from a single genomic strand and were enriched for non-retroviral endogenous viral elements (nrEVEs) in antisense orientation. Biogenesis of cluster-derived piRNAs depended on key factors, the mRNA exporter Nxf1, three Tudor proteins, Veneno, Tejas, and Yb, as well as the evolutionarily conserved cochaperone Shutdown. Combined transcriptional and chromatin state analyses showed that these core piRNA clusters do not have independent promoters, but instead depended on readthrough transcription from upstream genes for piRNA biogenesis. This biogenesis mechanism was evolutionarily conserved in other vector mosquito species, Aedes albopictus, Culex quinquefasciatus, and Anopheles gambiae. Interestingly, the upstream genes of many core piRNA clusters of the two Aedes mosquitoes were orthologous, but the downstream piRNA clusters differed both in size and sequence, especially evident by distinct nrEVE insertions from different virus families. We propose a model in which a conserved set of genes determine the loci for piRNA biogenesis, while the downstream piRNA clusters function as traps for nrEVEs, allowing adaptation to environmental challenges such as virus infection. Our systematic analyses of piRNA cluster expression and chromatin state lay a solid foundation for future studies of piRNA and genome evolution in these important vector species.

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