Genetic and mechanistic diversity of piRNA 3'-end formation

Abstract

Small regulatory RNAs guide Argonaute (Ago) proteins in a sequence-specific manner to their targets and therefore have important roles in eukaryotic gene silencing. 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 precursors. piRNA 5' ends are defined either by Zucchini, the Drosophila homologue of mitoPLD-a mitochondria-anchored endonuclease, or by piRNA-guided target cleavage. 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' ends, Aub/Ago3-mediated cleavages liberate pre-piRNAs that require extensive resection by the 3'-to-5' exoribonuclease Nibbler (Drosophila homologue of Mut-7). 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.

Figure ED1. 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) based on the piRNA loss (threshold = 5x loss). b) Boxplots 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).

Figure ED2. 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 germline. 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 Fig. ED3d. 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 Fig. ED2f. 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 color 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.

Figure ED3. 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 in percent (mean lengths are indicated below).

Figure ED4. A small RNA library cloning approach that allows the recovery of longer piRNA species.

Drosophila total RNA contains large amounts of the 31-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 the 2S rRNA depletion method published by Seitz et al. (2008), 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.

Figure ED5. Zucchini and Nibbler generate Aub- and Ago3-bound piRNA 3' ends independently of piRNA 5' end formation.

a) Boxplots (*** 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 10 equally sized bins, sorted for their 3' end precision index (see Methods). c) Boxplots (*** 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).

Figure ED6. 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) displays piRNAs bound to Piwi, Aubergine, or Ago3, while (b) displays piRNAs from total ovarian RNA. c) Plotted are 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). Boxplot indicates Aub/Ago3-bound piRNA levels in wild-type ovaries for defined TE groups (Tukey definition; *** p < 0.001 after Wilcoxon rank-sum test).

Figure ED7. 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 loose piRNAs only in Zucchini/Nibbler-depleted ovaries. b) Shown are 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).

Figure ED8. Precise coupling of neighboring 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 serve therefore as control. b) Length profiles (fractions of reads per indicated length in percent) of TE-mapping piRNAs bound to Aub/Ago3 in indicated genotypes. c) Boxplot 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 eleven germline enriched TEs that maintain piRNA production in Zucchini/Nibbler-depleted ovaries (see Methods). 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.

Figure ED9. piRNAs generated by slicer/slicer are methylated.

a) Shown are the mappings of those piRNAs (5' ends in black; 3' ends in gray) that were probed by Northern blots in Fig. 4f. The 5' ends of antisense piRNAs (red) map precisely 10nt 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.

Figure ED10. 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 orthologs 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).

Figure 1. The 3'-to-5' exonuclease Nibbler matures piRNA 3' ends from slicer-cleaved pre-piRNAs.

a) Schematic illustration of piRNA 5' and 3' biogenesis. b) Northern blot against individual piRNA 5' species (mature piRNAs: 23-29 nt) detects a 34-nt pre-piRNA (blue arrowhead). 3' end methylation probed by β-elimination (miR-8 serves as control). To the right, sequencing counts of the corresponding piRNAs (normalized to 1 million miRNA reads: ppm) from total small RNAs or from an Aub-IP are shown. c) Schematic of the dual-site piRNA biogenesis reporter. d) Levels of small RNAs (ppm) mapping to the biogenesis reporter (5' ends only) in indicated genetic backgrounds. e) Length profiles of TE-mapping small RNAs isolated from Piwi/Aub/Ago3-immuno-precipitates (genetic background indicated; p-values: two-sided t-test). f) Confocal images showing localization of GFP-Nibbler, Aub and Ago3 in w¹¹¹⁸ or aubergine mutant egg chambers (scale bar: 5 μm; individual channels as inverted gray scale images).

Figure 2. Two genetically independent pathways generate piRNA 3' ends.

a, b) Northern blot analysis (loading control: miR-8) and sequencing counts for an Ago3- (a) or an Aub- (b) enriched piRNAs in indicated genetic backgrounds. Bar charts display fraction of reads with indicated length. Respective RNA sequences are shown. c) Sequence logos display nucleotide bias around dominant 3' ends of Piwi/Aub/Ago3-bound piRNAs isolated from ovaries of indicated genetic background. Bar plots display the nucleotide composition at the position following dominant 3' ends (piRNAs are split into 10 equally sized bins sorted for their precision index). d) Shown is how the Nibbler/Zucchini contribution to 3' end formation of individual piRNA 5' species was determined. e) Shown are the Zucchini/Nibbler contributions for 3' end formation for Aub- and Ago3-bound piRNAs (left: as stacked bar diagram; right: as boxplot; Tukey definition; *** p < 0.001 after Wilcoxon rank-sum test).

Figure 3. Nibbler and Zucchini set the balance between primary and secondary piRNA biogenesis.

a) Schematic of the two-pathway model for piRNA 3' end formation. Panels b-c show scatter plots based on piRNAs mapping to germline-dominant TEs (n=63). b) Plotted are Z-scores of piRNA 3'/5' coupling in w¹¹¹⁸ versus nibbler^-/- ovaries (orange: Aub-bound antisense piRNA 3' ends to Piwi-bound piRNA 5' ends; blue: Ago3-bound sense piRNA 3' ends to Piwi-bound piRNA 5' ends). c) Plotted are normalized contributions of Piwi/Aub/Ago3-bound piRNAs to total TE piRNA profiles in w¹¹¹⁸ versus nibbler^-/- ovaries. Stacked bar plots show sum for all analyzed TEs.

Figure 4. An Argonaute-only pathway generates piRNAs in the absence of Zucchini and Nibbler.

a) Normalized 5' and 3' end counts (ppm) of piRNAs isolated from ovaries with indicated genetic background mapping to a ~100-nt sequence of Doc. b) Normalized 5' and 3' end counts (ppm) of piRNAs from Zucchini/Nibbler-depleted ovaries mapping to a biogenesis reporter with two (top) or three (bottom) piRNA target sites. c) Boxplots displaying Z-score distributions of 3'/5' coupling and 3'/5' ping-pong for piRNAs from ovaries of indicated genotype (n=11 germline TEs that maintain piRNA production in Zucchini/Nibbler-depleted ovaries). d, e) Displayed are 5' and 3' ends of piRNAs—isolated from Zucchini/Nibbler-depleted (d) or from Zucchini-depleted (e) ovaries—that map within a 40-nt window surrounding ping-pong responder piRNA 5' ends (position 0). Heat maps sorted first by dominant responder piRNA length and then by abundance. The sum of all 3' ends and all 5' ends per line is 100%. f) Northern blot analysis of piRNAs with dominant length of 21, 26, or 32 nt in Zucchini/Nibbler-depleted ovaries.