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. 2015 May;21(5):911-22.
doi: 10.1261/rna.048603.114. Epub 2015 Mar 23.

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

David Rosenkranz  1 , Stefanie Rudloff  1 , Katharina Bastuck  1 , René F Ketting  2 , Hans Zischler  1
Affiliations

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

David Rosenkranz et al. RNA. 2015 May.

Abstract

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.

Keywords: RNA interference; evolution; piRNA; siRNA; transposon defense.

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Figures

FIGURE 1.
FIGURE 1.
Analyses of testis-expressed small RNAs from the Northern Tupaia. (A) The Northern Tupaia (Tupaia belangeri, Zoo Frankfurt). (B) Phylogenetic relationship of the tupaia and presence/absence patterns of Piwi proteins in selected species. (C) Length profile and sequence annotation. (D) 5′ base composition of different small RNA fractions. (E) Ping-pong signature: preference for a 10 bp overlap of different sRNA fractions. (F) Interspersed 18–25 nt sequences do not show a ping-pong signature but tend to overlap with 18–22 nt. (E,F) Sequence counts refer to nonidentical sequences normalized by the number of genomic hits produced by each sequence.
FIGURE 2.
FIGURE 2.
Characteristics of tupaia piRNA clusters. (A) Example of a bidirectional piRNA cluster (proTRAC output, modified). Binding sites for A-MYB and RFX4 transcription factors are indicated (top). Most sequence reads map to unique loci (bottom). (B) TE content and composition of piRNA clusters compared with the whole genome. (C) TE-related piRNAs in piRNA clusters are biased toward antisense orientation. Dispersed TE-related sequences are biased toward sense orientation. (D) Location of predicted RFX4 and A-Myb binding sites relative to the center of bidirectional piRNA clusters. (E) Length profile of sense/antisense Tu-SINE2/3- and LINE1-related piRNAs.
FIGURE 3.
FIGURE 3.
Conservation of piRNA clusters in tupaia, mouse, and human. (A) Conserved piRNA cluster. Aligned blocks are indicated. Gray blocks in tupaia indicate probably artificial gaps filled with N's in the tupaia genome assembly. (B) Different degrees of sequence conservation for different residue classes within piRNA clusters. Values are presented for all pairwise piRNA cluster alignments (left) and those segments of tupaia piRNA clusters that could be aligned to both mouse and human (right). (C) Accumulation of selected lineage-specific TEs in genomes (dark blue) and piRNA clusters (light blue) over time. For information, the total TE content for genomes and piRNA clusters is indicated for each species.
FIGURE 4.
FIGURE 4.
Identification of sRNA source/target sites. (A) MariN1 transcripts form double-stranded siRNA precursor molecules that are most likely processed by Dicer resulting in typical duplexes with 2 nt 3′ overhangs. (B) Sequence composition of piRNAs and unclassified RNAs with respect to different TE classes. (C) Distribution of mapped sense and antisense sRNA reads along the TUS consensus sequence. Sequences were mapped allowing up to three mismatches. Black chart: amount of predicted paired bases in a 20 bp sliding window. (D) Predicted amount of paired bases at loci targeted by piRNAs and other sRNAs. 58.6% represent the average amount of predicted paired bases for all analyzed transposon transcripts. Dashed lines indicate the average amount of paired bases for sense and antisense targeted sites. Black lines indicate the standard deviation from the average value of 100 bootstrap pseudoreplicate-data sets. (E) TUS secondary structure (mfold, Zuker et al. 2003) visualized with PSEUDOVIEWER2 (Han and Byun 2003). Antisense-piRNA target site is highlighted in red.
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