Tupaia small RNAs provide insights into function and evolution of RNAi-based transposon defense in mammals.

Argonaute proteins comprising Piwi-like and Argonaute-like proteins and their guiding small RNAs combat mobile DNA on the transcriptional and post-transcriptional level. While Piwi-like proteins and associated piRNAs are generally restricted to the germline, Argonaute-like proteins and siRNAs have been linked with transposon control in the germline as well as in the soma. Intriguingly, evolution has realized distinct Argonaute subfunctionalization patterns in different species but our knowledge about mammalian RNA interference pathways relies mainly on findings from the mouse model. However, mice differ from other mammals by absence of functional Piwil3 and expression of an oocyte-specific Dicer isoform. Thus, studies beyond the mouse model are required for a thorough understanding of function and evolution of mammalian RNA interference pathways. We high-throughput sequenced small RNAs from the male Tupaia belangeri germline, which represents a close outgroup to primates, hence phylogenetically links mice with humans. We identified transposon-derived piRNAs as well as siRNAs clearly contrasting the separation of piRNA- and siRNA-pathways into male and female germline as seen in mice. Genome-wide analysis of tree shrew transposons reveal that putative siRNAs map to transposon sites that form foldback secondary structures thus representing suitable Dicer substrates. In contrast piRNAs target transposon sites that remain accessible. With this we provide a basic mechanistic explanation how secondary structure of transposon transcripts influences piRNA- and siRNA-pathway utilization. Finally, our analyses of tree shrew piRNA clusters indicate A-Myb and the testis-expressed transcription factor RFX4 to be involved in the transcriptional regulation of mammalian piRNA clusters.