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Genetic and mechanistic diversity of piRNA 3′-end formation

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Abstract

Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and therefore have important roles in eukaryotic gene silencing1. Of the three small RNA classes, microRNAs and short interfering RNAs are processed from double-stranded precursors into defined 21- to 23-mers by Dicer, an endoribonuclease with intrinsic ruler function. PIWI-interacting RNAs (piRNAs)—the 22–30-nt-long guides for PIWI-clade Ago proteins that silence transposons in animal gonads—are generated independently of Dicer from single-stranded precursors2,3. piRNA 5′ ends are defined either by Zucchini, the Drosophila homologue of mitoPLD—a mitochondria-anchored endonuclease4,5, or by piRNA-guided target cleavage6,7. Formation of piRNA 3′ ends is poorly understood. Here we report that two genetically and mechanistically distinct pathways generate piRNA 3′ ends in Drosophila. The initiating nucleases are either Zucchini or the PIWI-clade proteins Aubergine (Aub) or Ago3. While Zucchini-mediated cleavages directly define mature piRNA 3′ ends8,9, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3′-to-5′ exoribonuclease Nibbler (Drosophila homologue of Mut-7)10,11,12,13. The relative activity of these two pathways dictates the extent to which piRNAs are directed to cytoplasmic or nuclear PIWI-clade proteins and thereby sets the balance between post-transcriptional and transcriptional silencing. Notably, loss of both Zucchini and Nibbler reveals a minimal, Argonaute-driven small RNA biogenesis pathway in which piRNA 5′ and 3′ ends are directly produced by closely spaced Aub/Ago3-mediated cleavage events. Our data reveal a coherent model for piRNA biogenesis, and should aid the mechanistic dissection of the processes that govern piRNA 3′-end formation.

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Figure 1: The 3′-to-5′ exonuclease Nibbler matures piRNA 3′ ends from slicer-cleaved pre-piRNAs.
Figure 2: Two genetically independent pathways generate piRNA 3′ ends.
Figure 3: Nibbler and Zucchini set the balance between primary and secondary piRNA biogenesis.
Figure 4: An Argonaute-only pathway generates piRNAs in the absence of Zucchini and Nibbler.

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Acknowledgements

We thank all laboratory members for help and discussions, P. Duchek, J. Gokcezade and K. Meixner for generating fly lines, M. Novatchkova for help on the conservation analysis of nucleases, the VBCF NGS facility for sequencing, and the MFPL monoclonal facility for the Nibbler antibody. This work was supported by the Austrian Academy of Sciences, the European Community’s 7th Framework Program (ERC-StG-260711; ERC-StG-338252), the Austrian Science Fund (Y510-B12; F4303-B09; W12-7-B09; Y733-B22), and an HFSP postdoctoral fellowship to F.M.

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Authors and Affiliations

Authors

Contributions

F.M. made the initial observation that Nibbler trims Ago3-bound piRNAs, J.S. and R.H. did all experiments and did the computational analysis with the help of D.H. All authors designed the experiments and wrote the paper.

Corresponding authors

Correspondence to Stefan L. Ameres or Julius Brennecke.

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The authors declare no competing financial interests.

Additional information

Reviewer Information Nature thanks B. Czech and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 3′ ends of Zucchini-independent ping-pong piRNAs are formed by an exonuclease.

a, Scatter plot showing the fold change in piRNA levels (for 63 germline-dominant TEs) in Zucchini-depleted compared to control ovaries (calculated as sum of normalized Piwi/Aub/Ago3-bound piRNAs). TEs were grouped into robust (red) and sensitive (blue) on the basis of piRNA loss (threshold = 5× loss). b, Box plots displaying the Z-scores of canonical 5′/5′ ping-pong, 3′/5′ coupling, and 3′/5′ ping-pong for piRNAs isolated from ovaries of indicated genotype (for the 19 robust germline-enriched TEs that maintain piRNA production in Zucchini-depleted ovaries; defined in panel a). Midline indicates the median value, box ranges from the first to the third quartile, whiskers are 1.5× the interquartile range.

Extended Data Figure 2 Nibbler, but not Papi, is required for the generation of piRNAs in the absence of Zucchini.

a, Design of the short hairpin expression cassette that allows the simultaneous RNAi-mediated knockdown of two genes in a tissue specific manner. shRNAs are separated by the miR-6 backbone and can be cloned via indicated restriction sites. b, c, Confocal sections of egg chambers (scale bars, 10 μm) of indicated genotype expressing GFP-tagged Zucchini (b, c) and GFP-tagged Papi (b), showing the efficient shRNA-mediated knockdown of Zucchini and Papi (b), or Zucchini and Nibbler (c) in the germ line. Nibbler was detected using a monoclonal antibody. d, Confocal images showing the localization of GFP-tagged Papi and Nibbler in Drosophila egg-chambers (scale bars, 10 μm). Functionality of GFP–Nibbler is demonstrated in Extended Data Fig. 3d. e, Confocal sections through single nurse cell nuclei of egg chambers expressing GFP-tagged Zucchini (left) or Papi (right) and stained for mitochondria (immuno-staining of ATP synthase). f, Shown are mappings of piRNAs (5′ ends only; red, sense; black, antisense) from ovaries of indicated genotype to a second reporter construct (as in Fig. 1d). Values are normalized to 1 million sequenced miRNA reads. g, Scatter plot displaying miRNA-normalized piRNA levels mapping to 63 germline-dominant TEs in Zucchini-depleted versus Zucchini/Papi-depleted ovaries. h, Shown are confocal sections of egg chambers expressing indicated piRNA biogenesis reporters (GFP-fluorescence; DNA stained with DAPI) in wild-type (top) or Zucchini and Nibbler-depleted ovaries (middle and bottom). Top, reporter with no target site; middle, reporter used in Fig. 1d; bottom, reporter used in Extended Data Fig. 2f. i, Ovary lysates expressing N-terminally Flag-tagged wild-type Nibbler was immunoprecipitated (IPed) using M2 magnetic beads. Wild-type ovary lysates were used as a control. Red colour in the blot indicates a saturated signal. ATP synthase 5A (ATP syn) serves as loading control. Ovary volume indicates the amount of loaded lysate/IP fraction. j, Eluates from IP were blotted with indicated antibodies. Piwi is slightly enriched in the Flag–Nibbler IP fraction, while there is no detectable enrichment of Aub or Ago3 in the IP fraction.

Extended Data Figure 3 Molecular characterization of the piRNA pathway in nibbler and papi mutant flies.

a, papi gene locus indicating the position of the Cas9-induced frameshift allele. b, nibbler gene locus indicating the position of the Cas9-induced frameshift allele. c, Western blot analysis showing the loss of detectable Nibbler protein in nibbler−/− ovaries. ATP-synthase 5A antibody is used as loading control. Loaded amounts of ovary lysates are indicated. d, Northern blot analysis of the Nibbler-substrate miR-34 comparing small RNAs obtained from ovaries of w1118 or nibbler−/− flies. The GFP–Nibbler rescue transgene (used in Fig. 1f) restores miR-34 processing. e, f, Immunostainings (e) and western blot analysis (f) of Piwi, Aub and Ago3 in w1118 or in nibbler−/− ovaries, showing that localization and expression of the three PIWI-clade proteins are unperturbed (arrow heads; scale bars, 10 μm). ATP synthase 5A (ATP syn) served as loading control. g, Scatter plot displaying the steady-state RNA level of TEs in indicated genetic background (only TEs with RPKM >1 in either background; n = 40). h, Bar chart displaying TE mapping piRNA levels in w1118 or in nibbler−/− ovaries (values normalized to 1 million sequenced miRNA reads). i, j, Length profiles of TE mapping small RNA reads obtained from ovaries of indicated genotypes. Shown are all ovarian small RNAs (i) or Piwi-bound piRNAs defined as soma-enriched (j; see Methods). Displayed are fractions of reads of indicated length as a percentage (mean lengths are indicated below).

Extended Data Figure 4 A small RNA library cloning approach that allows the recovery of longer piRNA species.

Drosophila total RNA contains large amounts of the 30-nt long 2S rRNA. Previous cloning approaches therefore typically restrict small RNA cloning to the 18–29 nt window by cutting these small RNA populations from a gel. We used a previously published 2S rRNA depletion method35, followed by extracting small RNAs ranging from 18–40 nt in length for library preparation. Shown are size distributions of TE mapping small RNAs (obtained from w1118 ovaries) comparing the standard small RNA cloning protocol (left) and the protocol using total RNA depleted of 2S rRNA (middle; see Methods). An overlay of the longer reads (>27 nt) is displayed to the right.

Extended Data Figure 5 Zucchini and Nibbler generate Aub- and Ago3-bound piRNA 3′ ends independently of piRNA 5′-end formation.

a, Box plots (***P < 0.001 by two-sided t-test) showing the 3′-end definition (see Methods) of Ago3-, Aub-, and Piwi-bound piRNAs isolated from ovaries of the indicated genotypes. Soma-enriched Piwi-bound piRNAs (see Methods) are shown in boxes with diagonal lines. b, Stacked bar plots displaying the nucleotide composition at the 5′ end or position 10 of piRNAs bound to Aub-, Ago3-, or Piwi (isolated from ovaries of indicated genotypes). The plots show the composition within ten equally sized bins, sorted for their 3′ end precision index (see Methods). c, Box plots (***P < 0.001 by Wilcoxon rank-sum test) showing the R2 values of the comparison between the 3′-end profiles of Ago3- or Aub-bound piRNAs from w1118 ovaries and those of nibbler−/− ovaries (Zucchini only), those of ovaries depleted of Zucchini (Nibbler only), and those of the calculated composite that provides the highest R2 (best fit). Midlines in a and c indicate the median value, box ranges from the first to the third quartile, whiskers are 1.5× the interquartile range

Extended Data Figure 6 Ago3 incorporates more TE antisense piRNAs in nibbler mutant flies.

a, b, Scatter plots displaying the strand bias (antisense divided by sense) of piRNA populations in w1118 versus nibbler−/− ovaries. a, piRNAs bound to Piwi, Aub, or Ago3. b, piRNAs from total ovarian RNA. c, Z-scores of 5′/5′ ping-pong levels per TE (63 germline dominant TEs) in w1118 versus nibbler−/− ovaries. d, Grouping of TEs (63 germline dominant TEs) based on fold change in piRNA levels between Zucchini-depleted and control ovaries (left). Box plot indicates Aub/Ago3-bound piRNA levels in wild-type ovaries for defined TE groups (midline indicates the median value, box the first and third quartiles, whiskers are 1.5× the interquartile range; ***P < 0.001 after Wilcoxon rank-sum test).

Extended Data Figure 7 piRNAs are abundantly produced in Zucchini/Nibbler double-depleted ovaries.

a, Heat map displaying fold changes in antisense piRNA levels per TE (n = 63) in indicated genotypes versus control (six biological replicates). Dots mark TEs, which lose piRNAs only in Zucchini/Nibbler-depleted ovaries. b, Mappings (5′ ends only; red, sense; black, antisense) of piRNAs onto the GATE and Doc TE consensus sequences. The plots at the top are from a piRNA library obtained from Zucchini-depleted ovaries, and the ones at the bottom are from a piRNA library obtained from Zucchini/Nibbler double-depleted ovaries. The ~100-bp window of Doc that is detailed in Fig. 4a is depicted by brackets. c, Size distributions of TE-mapping small RNAs obtained from total small RNA libraries from Zucchini-depleted (top) or Zucchini/Nibbler-depleted (bottom) ovaries. Shown are the average values from six biological replicates (reads were normalized to 1 million sequenced miRNA reads). d, Scatter plots displaying the steady-state RNA level of TEs in indicated genetic background (only TEs with RPKM >1 in either background are shown; n = 74).

Extended Data Figure 8 Precise coupling of neighbouring ping-pong piRNAs in Zucchini/Nibbler-depleted ovaries.

a, Immunofluorescence images (confocal sections) of egg chambers of indicated genotype stained for endogenous Piwi protein (scale bars, 10 μm). Note that the shRNA-mediated knockdown is specific for germline cells. Somatic follicle cells that also express Piwi therefore serve as control. b, Length profiles (fractions of reads per indicated length as a percentage) of TE-mapping piRNAs bound to Aub/Ago3 in indicated genotypes. c, Box plot (midline indicates the median value, box the first and third quartiles, whiskers are 1.5× the interquartile range) displaying the distribution of Z-scores for canonical 5′/5′ ping-pong of piRNA populations isolated from ovaries of indicated genotypes. The analysis is restricted to the 11 germline enriched TEs that maintain piRNA production in Zucchini/Nibbler-depleted ovaries (see Methods). Midline indicates the median value, box ranges from the first to the third quartile, whiskers are 1.5× the interquartile range. d, Histogram showing the frequencies of a cloned ping-pong piRNA 5′ end downstream of a responder piRNA 5′ end at a certain distance on the same strand. e, Sequence logos displaying the nucleotide composition within and downstream of Aub- and Ago3-bound piRNAs cloned from ovaries of indicated genotypes. 5′-end position and position 10 are measured from the piRNA 5′ end. 3′-end position is the dominant 3′ end of a certain piRNA 5′ species. Downstream positions are anchored by the dominant 3′-end position. Nucleotide signatures with respect to the position of sense and antisense slicer piRNAs are depicted in the cartoons below.

Extended Data Figure 9 piRNAs generated by slicer/slicer are methylated.

a, Shown are the mappings of those piRNAs (5′ ends in black; 3′ ends in grey) that were probed by northern blots in Fig. 4f. The 5′ ends of antisense piRNAs (red) map precisely 10 nt downstream of the predicted slicer sites. Mappings were normalized to 1 million sequenced miRNA reads. b, An oxidized library control of the heat map shown in Fig. 4d, showing the precise coupling of piRNA 5′ ends (blue) and 3′ ends (red) in Zucchini/Nibbler-depleted ovaries.

Extended Data Figure 10 Conservation of Zucchini/MitoPLD, PARN, PNLDC1, and Nibbler/Mut-7 across metazoa.

328 metazoan species are displayed in a phylogenetic tree (generated using iTOL). The presence of indicated orthologues in each species is marked (black, Zucchini/MitoPLD; green, PARN; blue, PNLDC1; red, Nibbler/Mut-7). Taxonomic groups mentioned in the text are highlighted (for details see Methods).

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Supplementary Information

This file contains the Source Data for Figures 1b, 2a,b, 4f and Extended Data Figures 2i, j, 3d, f as well as a Supplementary Table, which shows the high through datasets analyzed in the study. (PDF 2394 kb)

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Hayashi, R., Schnabl, J., Handler, D. et al. Genetic and mechanistic diversity of piRNA 3′-end formation. Nature 539, 588–592 (2016). https://doi.org/10.1038/nature20162

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