A role for transcription from a piRNA cluster in de novo piRNA production

Abstract

PIWI-interacting RNAs (piRNAs) are at the heart of the nucleic acid-based adaptive immune system against transposons in animal gonads. To date, how the piRNA pathway senses an element as a substrate and how de novo piRNA production is initiated remain elusive. Here, by utilizing a GFP transgene, we screened and obtained clonal silkworm BmN4 cell lines producing massively amplified GFP-derived piRNAs capable of silencing GFP in trans. In multiple independent cell lines where GFP expression was silenced by the piRNA pathway, we detected a common transcript from an endogenous piRNA cluster, in which a part of the cluster is uniquely fused with an antisense GFP sequence. Bioinformatic analyses suggest that the fusion transcript is a source of GFP primary piRNAs. Our data implicate a role for transcription from a piRNA cluster in initiating de novo piRNA production against a new insertion.

piRNA dynamics in divergent zebrafish strains reveal long-lasting maternal influence on zygotic piRNA profiles

“…Indeed, such flexibility has been documented in recent studies. First, attempts to generate transgenic silkworm cell lines showed a remarkable incidence of transgenes integrating into piRNA clusters, supporting the idea that piRNA clusters act as a sink for active transposable elements (Kawaoka et al 2012). Furthermore, studies in mouse and Drosophila showed that inserting GFP sequences in piRNA clusters resulted in GFP-derived piRNAs and stable silencing of GFP in trans (Muerdter et al 2012).…”

Hsp90 facilitates accurate loading of precursor piRNAs into PIWI proteins

“…In the current model for piRNA-RISC formation, single-stranded primary transcripts are first produced from piRNA-generating loci. Then, these transcripts are likely fragmented into precursor piRNAs (pre-piRNAs) that are longer than mature piRNAs, through multiple steps including endonucleolytic cleavage by Zucchini (Brennecke et al 2007;Klattenhoff et al 2009;Ipsaro et al 2012;Kawaoka et al 2012;Muerdter et al 2012;Nishimasu et al 2012). A subset of PIWI proteins (e.g., Siwi in the silkworm) preferentially binds to pre-piRNAs with 5 ′ U (1U) (Brennecke et al 2007;Gunawardane et al 2007; Kawaoka et al 2009).…”

Piwi Proteins and piRNAs Step onto the Systems Biology Stage

“…We know that piRNAs are longer in length than miRNAs and endo-siRNAs (at 25–31 nt), the 3' terminal 2' oxygen is methylated by the Hen1 enzyme, and there are many putative helicases, putative endonucleases, and tudor-domain containing proteins genetically required to generate and/or stabilize piRNAs (Ishizu et al 2012). However, our mechanistic picture of the functions for these Piwi pathway genes remains murky: the only in vitro piRNA biogenesis activity known is a “Trimmer” activity observed in Bombyx gonadal cell extracts which can trim a long 5' phosphorylated transcript bound by SIWI into a mature piRNA (Kawaoka et al 2012). And even though genetic advances that place artificial sequences like GFP into a piRNA precursor can give rise to artificial piRNAs (Kawaoka et al 2012; Muerdter et al 2012), we still cannot predict exactly which piRNAs would be generated most abundantly from these piRNA-generating transgenes.…”

“…However, our mechanistic picture of the functions for these Piwi pathway genes remains murky: the only in vitro piRNA biogenesis activity known is a “Trimmer” activity observed in Bombyx gonadal cell extracts which can trim a long 5' phosphorylated transcript bound by SIWI into a mature piRNA (Kawaoka et al 2012). And even though genetic advances that place artificial sequences like GFP into a piRNA precursor can give rise to artificial piRNAs (Kawaoka et al 2012; Muerdter et al 2012), we still cannot predict exactly which piRNAs would be generated most abundantly from these piRNA-generating transgenes.…”