Nature | Vol 578 | 13 February 2020 | 311Article
Zucchini consensus motifs determine the
mechanism of pre-piRNA production
Natsuko Izumi1,5, Keisuke Shoji1,2,5, Yutaka Suzuki^3 , Susumu Katsuma^4 & Yukihide Tomari1,3*PIWI-interacting RNAs (piRNAs) of between approximately 24 and 31 nucleotides in
length guide PIWI proteins to silence transposons in animal gonads, thereby ensuring
fertility^1. In the biogenesis of piRNAs, PIWI proteins are first loaded with
5′-monophosphorylated RNA fragments called pre-pre-piRNAs, which then undergo
endonucleolytic cleavage to produce pre-piRNAs^1 ,^2. Subsequently, the 3′-ends of pre-
piRNAs are trimmed by the exonuclease Trimmer (PNLDC1 in mouse)^3 –^6 and 2′-O-
methylated by the methyltransferase Hen1 (HENMT1 in mouse)^7 –^9 , generating mature
piRNAs. It is assumed that the endonuclease Zucchini (MitoPLD in mouse) is a major
enzyme catalysing the cleavage of pre-pre-piRNAs into pre-piRNAs^10 –^13. However,
direct evidence for this model is lacking, and how pre-piRNAs are generated remains
unclear. Here, to analyse pre-piRNA production, we established a Trimmer-knockout
silkworm cell line and derived a cell-free system that faithfully recapitulates Zucchini-
mediated cleavage of PIWI-loaded pre-pre-piRNAs. We found that pre-piRNAs are
generated by parallel Zucchini-dependent and -independent mechanisms. Cleavage
by Zucchini occurs at previously unrecognized consensus motifs on pre-pre-piRNAs,
requires the RNA helicase Armitage, and is accompanied by 2′-O-methylation of pre-
piRNAs. By contrast, slicing of pre-pre-piRNAs with weak Zucchini motifs is achieved
by downstream complementary piRNAs, producing pre-piRNAs without 2′-O-
methylation. Regardless of the endonucleolytic mechanism, pre-piRNAs are matured
by Trimmer and Hen1. Our findings highlight multiplexed processing of piRNA
precursors that supports robust and flexible piRNA biogenesis.piRNAs are a class of small RNAs, approximately 24−31 nucleotides (nt) in
size, produced from transposons and from discrete genomic loci called
piRNA clusters^14 , and guide PIWI proteins to target transcripts. PIWI
proteins possess an endonucleolytic activity, referred to as ‘slicer’, which
directly cleaves target RNAs in the cytoplasm^14 –^17. In addition, a subset of
PIWI proteins mediates transcriptional silencing in the nucleus^18 –^20. In
germ cells, piRNA biogenesis is coupled with reciprocal slicing between
complementary transcripts derived from transposons and piRNA clus-
ters, a process called the ping-pong cycle^14 ,^15 (Extended Data Fig. 1).
To generate mature piRNAs, PIWI proteins are first loaded with long
single-stranded RNA fragments bearing a 5′ monophosphate, called pre-
pre-piRNAs^1 ,^2. The pre-pre-piRNA is then endonucleolytically cleaved
at a position 3′ downstream of the PIWI-bound region to generate two
cleavage fragments^1 ,^2 ,^12 ,^13. In mice, silkworms and many other animals,
the 5′-cleavage fragment, called a pre-piRNA, is shortened to the mature
length by Trimmer (PNLDC1 in mouse), a PARN-like 3′-to-5′ exonuclease
localized on the mitochondrial surface^3 –^6 , and 2′-O-methylated by the
methyltransferase Hen1 (HENMT1 in mouse)^7 –^9. The 3′ cleavage fragment
is loaded into the next PIWI protein as a new pre-pre-piRNA. As a result,
a series of ‘trailing’ pre-piRNAs are consecutively generated^1 ,^2 ,^12 ,^13 and
matured by Trimmer and Hen1 (Extended Data Fig. 1).
The endonuclease Zucchini (MitoPLD or PLD6 in mouse)^10 ,^11 , which is
localized on the mitochondrial outer membrane, is assumed to mediate
cleavage of the PIWI-bound pre-pre-piRNAs^12 ,^13. Because trailing piRNAs
often start with a 5′ uridine (U)^2 ,^4 ,^12 ,^13 , it is believed that cleavage activity
of Zucchini has a preference for a site immediately before U in vivo.
However, purified Zucchini protein shows nonspecific endoribonucle-
ase activity in vitro^10 ,^11 ,^21. Owing to this discrepancy, the identity of the
endonuclease for pre-pre-piRNAs remains unclear and is ambiguously
and cautiously described in the literature^1 ,^2 ,^22. Thus, an in vitro system
that can faithfully recapitulate the endonucleolytic reaction mediated
by Zucchini is needed to resolve these ambiguities and discrepancies.Two parallel pathways produce pre-piRNAs
To accurately investigate how pre-piRNAs are generated from pre-pre-
piRNAs, it is necessary to block further processing of pre-piRNAs by
Trimmer. In previous studies, knockdown of Trimmer in the silkworm
cell line BmN4 resulted in only a slight extension of piRNA lengths^3 ,^21 ,
suggestive of residual Trimmer activity. To overcome this, we used
CRISPR–Cas9 to generate Trimmer-knockout (Tri-KO) BmN4 cells
(Extended Data Fig. 2a−c). We identified Tri-KO lines deficient in bothhttps://doi.org/10.1038/s41586-020-1966-9
Received: 6 July 2019
Accepted: 25 November 2019
Published online: 29 January 2020
(^1) Laboratory of RNA Function, Institute for Quantitative Biosciences, The University of Tokyo, Tokyo, Japan. (^2) Department of Agrobiology and Bioresources, School of Agriculture, Utsunomiya
University, Utsunomiya, Japan.^3 Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Kashiwa, Japan.^4 Department of
Agricultural and Environmental Biology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo, Japan.^5 These authors contributed equally: Natsuko Izumi,
Keisuke Shoji. *e-mail: [email protected]