shutdown is a component of the Drosophila piRNA biogenesis machinery

Abstract

In animals, the piRNA pathway preserves the integrity of gametic genomes, guarding them against the activity of mobile genetic elements. This innate immune mechanism relies on distinct genomic loci, termed piRNA clusters, to provide a molecular definition of transposons, enabling their discrimination from genes. piRNA clusters give rise to long, single-stranded precursors, which are processed into primary piRNAs through an unknown mechanism. These can engage in an adaptive amplification loop, the ping-pong cycle, to optimize the content of small RNA populations via the generation of secondary piRNAs. Many proteins have been ascribed functions in either primary biogenesis or the ping-pong cycle, though for the most part the molecular functions of proteins implicated in these pathways remain obscure. Here, we link shutdown (shu), a gene previously shown to be required for fertility in Drosophila, to the piRNA pathway. Analysis of knockdown phenotypes in both the germline and somatic compartments of the ovary demonstrate important roles for shutdown in both primary biogenesis and the ping-pong cycle. shutdown is a member of the FKBP family of immunophilins. Shu contains domains implicated in peptidyl-prolyl cis-trans isomerase activity and in the binding of HSP90-family chaperones, though the relevance of these domains to piRNA biogenesis is unknown.Keywords: piRNAs; transposon silencing; RNAi; FKBP; germ cells INTRODUCTIONEukaryotic genomes are prone to the accumulation of repetitive sequences, including transposable elements, over evolutionary time (McClintock 1953;Kim et al. 1994;Brennecke et al. 2007;Chambeyron et al. 2008;Feschotte 2008). The genomic instability brought about by transposon activity is a double-edged sword. Low levels of transposition can drive evolution in the long term, but loss of control over mobile elements in any individual can threaten reproductive success. Mechanisms for suppressing transposon activation in the germline are therefore both potent and widely conserved (Grimson et al. 2008). In animals, the PIWI-interacting RNA (piRNA) pathway is key to transposon silencing in reproductive tissues (Aravin et al. 2006;Girard et al. 2006;Lau et al. 2006;Vagin et al. 2006;Khurana and Theurkauf 2010;Senti and Brennecke 2010). In Drosophila, piRNAs are active both in the germ cell lineage and in a particular somatic lineage that encysts the germ cells and provides growth and maturation signals piRNA clusters sit at the apex of the pathway and, based upon their sequence content, define transposon targets for repression (Brennecke et al. 2007). piRNA clusters give rise to long, single-stranded transcripts (Brennecke et al. 2007) that are thought to be exported to the cytoplasm and processed into primary piRNAs, most likely in specialized cytoplasmic structures (Saito et al. 2010;Handler et al. 2011). A number of proteins have been implicated in primary piRNA biogenesis and their loading into PIWI-family proteins, including Armitage, Zucchini, Vreteno, and the Yb family (Klattenhoff et al...

The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing

“…6). Additional proteins, such as GASZ, Tdrds, and chaperones, are likely components of the pICL complex (Olivieri et al 2012;Preall et al 2012;Xiol et al 2012;Izumi et al 2013). MOV10L1 does not interact with Tdrkh (Saxe et al 2013), which is required for 3 ′ end maturation.…”

Multiple roles for Piwi in silencing Drosophila transposons

“…Small RNA cloning and library normalization Small RNA libraries used in the analysis were previously published (Preall et al 2012). However, in the present study, a different normalization strategy was used.…”

Hsp90 facilitates accurate loading of precursor piRNAs into PIWI proteins

“…Recent reports have implicated Heat shock protein 90 (Hsp90) and its cochaperones in the piRNA pathway (Specchia et al 2010;Gangaraju et al 2011;Olivieri et al 2012;Preall et al 2012;Xiol et al 2012). In flies, disruption of Hsp90 causes reduction of a subset of piRNAs and de-silencing of a number of transposons (Specchia et al 2010).…”

“…Hsp90 interacts with Piwi, one of the three fly PIWI proteins, and regulates the phosphorylation level of Piwi without affecting its protein stability (Gangaraju et al 2011). Moreover, an Hsp90 cochaperone, Shutdown, plays a critical role in both primary and secondary piRNA biogenesis in flies (Olivieri et al 2012;Preall et al 2012), while Fkbp6, a mouse ortholog of Shutdown, is exclusively required for secondary piRNA biogenesis (Xiol et al 2012). In the silkworm ovary-derived cell line BmN4, inhibition of Hsp90 causes accumulation of 16-nt RNAs in BmAgo3 (but not in Siwi), which presumably correspond to the 5 ′ fragments of Siwi-directed cleavage products (Xiol et al 2012).…”