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Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm

Nature volume 531, pages 390–394 (2016)Cite this article

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Abstract

The conserved Piwi family of proteins and piwi-interacting RNAs (piRNAs) have a central role in genomic stability, which is inextricably linked to germ-cell formation, by forming Piwi ribonucleoproteins (piRNPs) that silence transposable elements1. In Drosophila melanogaster and other animals, primordial germ-cell specification in the developing embryo is driven by maternal messenger RNAs and proteins that assemble into specialized messenger ribonucleoproteins (mRNPs) localized in the germ (pole) plasm at the posterior of the oocyte2,3. Maternal piRNPs, especially those loaded on the Piwi protein Aubergine (Aub), are transmitted to the germ plasm to initiate transposon silencing in the offspring germ line4,5,6,7. The transport of mRNAs to the oocyte by midoogenesis is an active, microtubule-dependent process8; mRNAs necessary for primordial germ-cell formation are enriched in the germ plasm at late oogenesis via a diffusion and entrapment mechanism, the molecular identity of which remains unknown8,9. Aub is a central component of germ granule RNPs, which house mRNAs in the germ plasm10,11,12, and interactions between Aub and Tudor are essential for the formation of germ granules13,14,15,16. Here we show that Aub-loaded piRNAs use partial base-pairing characteristics of Argonaute RNPs to bind mRNAs randomly in Drosophila, acting as an adhesive trap that captures mRNAs in the germ plasm, in a Tudor-dependent manner. Notably, germ plasm mRNAs in drosophilids are generally longer and more abundant than other mRNAs, suggesting that they provide more target sites for piRNAs to promote their preferential tethering in germ granules. Thus, complexes containing Tudor, Aub piRNPs and mRNAs couple piRNA inheritance with germline specification. Our findings reveal an unexpected function for piRNP complexes in mRNA trapping that may be generally relevant to the function of animal germ granules.

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Figure 1: Transcriptome-wide identification of RNAs bound by Aub and in vivo retrotransposon targeting and slicing captured by CLIP.
Figure 2: Complementarity analysis between the piRNA and mRNA parts of chimaeric CLIP tags.
Figure 3: Characteristics of piRNA base-pairing identified by chimaeric CLIP tag analysis.
Figure 4: Transcriptome-wide prediction of piRNA target sites and length differential of posterior-localized mRNAs.

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Sequence Read Archive

Data deposits

Sequences have been deposited in the Sequence Read Archive (SRA) under the accession number SRP067739.

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Acknowledgements

We thank former and current laboratory members for discussions; M. Siomi for the Tudor antibody; A. Arkov for tud flies; G. Dreyfuss for the PABP antibody; and J. Schug for Illumina sequencing. Work was supported by a Brody family fellowship to M.M., and a National Institutes of Health (NIH) grant GM072777 to Z.M.

Author information

Author notes

  1. Anastassios Vourekas and Panagiotis Alexiou: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pathology and Laboratory Medicine, Division of Neuropathology, Institute for Translational Medicine and Therapeutics, Perelman School of Medicine; PENN Genome Frontiers Institute, University of Pennsylvania,

    Anastassios Vourekas, Panagiotis Alexiou, Nicholas Vrettos, Manolis Maragkakis & Zissimos Mourelatos

  2. PENN Genome Frontiers Institute, University of Pennsylvania, Philadelphia, 19104, Pennsylvania, USA

    Anastassios Vourekas, Panagiotis Alexiou, Nicholas Vrettos, Manolis Maragkakis & Zissimos Mourelatos

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Contributions

A.V. and Z.M. conceived, and Z.M. supervised, the study. A.V. and N.V. performed the experiments. P.A. performed bioinformatic analyses with contribution from M.M. and A.V. A.V., P.A., N.V., M.M. and Z.M. interpreted the data. A.V. wrote the manuscript, with contribution from all authors.

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Correspondence to Zissimos Mourelatos.

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The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 Endogenous Aub localization in genotypes used, sequenced and mapped reads of CLIP sequencing and RNA immunoprecipitation libraries used in this study, and general characteristics of yw ovary and tud embryo (0–2 h) CLIP sequencing libraries.

a, Immunofluorescence of ovary and early embryo of indicated genotypes using antibodies against Aub (Aub-83; green) and Tudor (red), and schematic representation of the egg chamber. Aub is localized in the nuage and germ (pole) plasm of wild-type ovaries, in the germ plasm of early wild-type embryos (stage 2) and within PGCs as they form in the posterior pole (stage 5), and as they migrate during gastrulation (stage 10). Tudor colocalizes with Aub in the germ plasm of early embryos but is not detected after PGC formation. In Tudor mutant early embryos, Aub is not concentrated in the posterior but is diffusely present throughout the embryo; PGCs are never specified resulting in agametic adults (see also Extended Data Fig. 9). b, Sequenced and mapped reads of CLIP sequencing (CLIP-seq) libraries prepared in this study. c, Sequenced and mapped reads of RNA immunoprecipitation deep-sequencing libraries prepared in this study. d, Size distribution for the three low (one for tud) and three high yw ovary and tud embryo (0–2 h) Aub CLIP-seq libraries. The size range of piRNAs (23–29 nucleotides) is indicated by a dashed box. e, Average 5′ end nucleotide composition for piRNAs (23–29 nucleotides) from three low yw ovary, tud embryo (0–2 h) (one library) and yw embryo (0–2 h) Aub CLIP-seq libraries. f, Average 5′ end nucleotide composition of CLIP tags from three high yw ovary and tud embryo (0–2 h) Aub CLIP-seq libraries. piRNAs (23–29 nucleotides) are indicated by a dashed box. g, Genomic distribution of CLIP tags for three high yw ovary and tud embryo (0–2 h) Aub CLIP-seq libraries. Overlap of piRNAs from CLIP and immunoprecipitation libraries. All error bars denote s.d.; n = 3.

Extended Data Figure 2 Pairwise comparisons of transposon piRNA populations from various libraries.

a–c, Scatterplot comparison of normalized abundance of piRNAs mapped on consensus retrotransposon sequences (sense and antisense), from yw embryo (0–2 h) standard Aub immunoprecipitation and Aub CLIP libraries (a); from yw ovary libraries (b); and from tud embryo (0–2 h) libraries (c). Pearson correlation is shown for all elements in each plot. Retrotransposon categories are set as in ref. 31. d–f, Scatterplot comparison of normalized abundance of transposon-derived piRNAs in Aub CLIP libraries prepared from higher molecular mass signals (high; Fig. 1a, marked with a light blue line), with the piRNAs found in the libraries prepared from the main radioactive signal (low; Fig. 1a, marked with a dark blue line) from yw embryo (0–2 h) (d); from yw ovary Aub CLIP ‘high’ and ‘low’ libraries (e); and from tud embryo (0–2 h) Aub CLIP high and low libraries (f). These comparisons indicate that the piRNA loads in low and high CLIP libraries are essentially identical. g, Scatterplot comparison of normalized abundance of transposon-derived piRNAs for yw ovary and tud ovary Aub immunoprecipitation libraries, to evaluate changes of piRNA load in the absence of Tudor. While antisense-derived piRNAs are largely unchanged, a few sense-derived piRNAs are changed (blood retrotransposon is indicated). h, i, Scatterplot comparison of normalized abundance of transposon-derived piRNAs for yw ovary and yw embryo (0–2 h) Aub immunoprecipitation libraries (h); and for tud ovary and tud embryo (0–2 h) libraries (i).

Extended Data Figure 3 Retrotransposon targeting by complementary piRNAs identified by Aub CLIP.

a, Overlap of lgCLIPs with complementary piRNAs from CLIP libraries, mapping on retrotransposons. b, c, Scatterplots of normalized abundance of antisense piRNAs and sense lgCLIPs (b) and for sense piRNAs and antisense lgCLIPs (c) mapped on retrotransposons for the indicated Aub CLIP libraries. Pearson correlation is shown for all elements in every plot. Retrotransposon categories are set as in ref. 31.

Extended Data Figure 4 CLIP identifies extensive mRNA binding by Aub.

a, Ratio average plot of normalized (reads per million, RPM) Aub CLIP tag (pi, piRNA; lg, lgCLIP) abundance (A value) versus lgCLIPs over piRNA abundance (R value), for all mRNAs. Outlined circles (red) correspond to genes that belong in the 12 posterior localization categories depleted in tud versus yw Aub CLIP libraries. Zero values are substituted with a small (smallest than the minimum) value so that log calculations are possible. This graph strongly suggests that mRNA binding by Aub as captured by CLIP is not for piRNA biogenesis purposes. b, Sequenced and mapped reads of RNA-seq libraries prepared in this study. c, Density of Aub CLIP-seq tags (yw embryo, and bottom panel: tud embryo) and RNA-seq reads (top panel: yw embryo) within the UTRs and coding sequences of the meta-mRNA. Each mRNA region is divided in 30 bins, and the number of the chimaeric mRNA fragments (genomic coordinate of the mRNA fragment midpoint) mapped within each bin is counted. Error bars indicate one s.d., n = 3 for CLIP-seq; minimum and maximum values for the two RNA-seq replicate libraries. d, Scatterplot of average normalized mRNA abundance for yw embryo RNA-seq (RPKM) and Aub CLIP-seq (RPM). Aub highly bound mRNAs with posterior localizations (Supplementary Table 4) are marked with a red circle. Zero values are substituted with a small (smallest than the minimum) value so that log calculations are possible. CLIP-seq identifies mRNAs that span the whole expression range of RNA-seq libraries, indicating that Aub CLIP does not capture transcripts simply based on abundance.

Extended Data Figure 5 Partial purification of Aub RNPs from early embryo supports binding of germ plasm mRNAs by Aub.

a, Fractionation of isopycnic Nycodenz density gradients of post-nuclear yw embryo lysate. Protein and Nycodenz concentration for every fraction is plotted. b, Western blot detection of indicated proteins in gradient fractions. A short and a long exposure (exp.) for Aub is shown. Uncropped gels for b, d and e can be found in Supplementary Fig. 1. c, Heat map of levels of indicated germ plasm mRNAs determined by quantitative RT–PCR (qRT–PCR), normalized to spiked luciferase RNA, and with fraction 2 as a reference. d, Western blot detection of Aub in indicated diluted Nycodenz fractions used for Aub RNA immunoprecipitation. e, Electrophoretic analysis on denaturing polyacrylamide gel of 32P-labelled small RNAs immunoprecipitated with Aub from indicated gradient fractions. A bracket denotes piRNAs, detected primarily in fractions 6 and 7 (asterisk denotes 2S rRNA). f, Bar chart showing fold enrichment (over fraction-extracted total RNA) of indicated germ plasm mRNAs in Aub immunoprecipitations from gradient fractions, measured by qRT–PCR. Luciferase mRNA was used as a spike.

Extended Data Figure 6 Analysis of Aub CLIP tags mapping to mRNAs with regard to the presence of mRNA embedded transposons.

a, Overlap of lgCLIPs with complementary piRNAs from CLIP libraries, mapping on mRNAs. b, Scatterplot of yw embryo Aub lgCLIPs mapped in the sense orientation on mRNAs, with piRNAs mapped in the antisense orientation. Zero values are substituted with a small (smallest than the minimum) value so that log calculations are possible. Contrary to retrotransposons (Extended Data Fig. 3), there is no correlation, suggesting that extensive piRNA complementarity cannot explain the widespread mRNA binding shown by mRNA lgCLIPs. c, Scatterplot of yw embryo Aub lgCLIPs mapped in the sense orientation on mRNAs with per base (nucleotide) mRNA embedded retrotransposons (LINE, long terminal repeat (LTR), satellite). Posterior, non-posterior and undetermined localizations are marked as indicated. The graph is separated into four quadrants: clockwise from bottom left corner: 0 embedded repeats, 0 CLIP tags; 0 embedded repeats, >0 CLIP tags; >0 embedded repeats, >0 CLIP tags, >0 embedded repeats, 0 CLIP tags. The number of genes in the four quadrants is indicated. Zero values are substituted with a small (smallest than the minimum) value (different small value for every localization category was used for clarity) so that log calculations are possible. This graph suggests that there is no correlation between the numbers of CLIP tags and embedded repeats within the mRNAs. d, Aub lgCLIPs density surrounding (±200 bases) mRNA-embedded retrotransposons (LINE, LTR, satellite as indicated). This analysis shows that there is no increase in the lgCLIP density in the areas flanking embedded repeats, suggesting that repeat sequences are not used as enriched target areas for mRNA binding by Aub. Error bars denote s.d.; n = 3. e, Analysis of mRNA expression level in relation to the number of embedded repeats. The number of embedded repeats per nucleotide of exon was plotted with the ratio (log10) of mRNA expression in yw embryo (0–2 h) versus aubHN2/QC42 embryo (0–2 h) (left), and yw embryo (0–2 h) versus tud embryo (0–2 h) (right). The mRNAs are divided into groups based on the number of embedded repeats. The number above each data point denotes the number of mRNAs in each group. The graphs suggest that there is no proportional or consistent abundance change, decrease or increase, with the number of embedded repeats.

Extended Data Figure 7 Characteristics of piRNAs and piRNA base-pairing with complementary target sites identified from analysis of chimaeric CLIP tags.

a, piRNA–mRNA complementarity events for a random piRNA (negative control, average of three yw (top) and tud (bottom) embryo (0–2 h) samples), within ±100 bases from the midpoint of the mRNA part of the chimaeric read. Complementarity events are plotted per alignment score group as indicated, for clarity. Inset (per sample): bar chart of average complementarity events per score group. b, Size distribution of the piRNAs identified within chimaeric CLIP tags, for yw and tud embryo CLIP libraries. Only the piRNAs implicated in the complementarity events occurring within ±25 nucleotides from the midpoint of the mRNA fragment and with score ≥7 are analysed in this graph, and the graphs in c–e, g–i. c, 5′ end nucleotide preference for the piRNAs identified within chimaeric CLIP tags, for yw and tud embryo Aub CLIP libraries. d, Genomic distribution for the piRNAs identified within chimaeric CLIP tags, for yw and tud embryo Aub CLIP libraries. e, Per position nucleotide preference for all piRNAs in Aub yw embryo (0–2 h) CLIP library L3 (left), and for the piRNAs identified within chimaeric CLIP tags, for yw and tud embryo Aub CLIP libraries. f, Complementarity events between piRNAs and mRNA fragments of chimaeric reads, for posterior and non-posterior localized mRNAs (yw embryo). The plots are separated per score group. g, Heat maps showing base-paired nucleotides of piRNAs for all complementarity events identified within chimaeric CLIP tags (events occurring within ±25 nucleotides from mRNA fragment midpoint, score ≥7) for tud embryo. Colour is according to the length of the consecutive stretch of base-paired nucleotides that runs over every position (colour code shown on the right). Stacked piRNAs are aligned at their 5′ ends and sorted (bottom to top) following these rules: (a) starting position of the longest stretch of consecutive base paired nucleotides, relative to the piRNA end; (b) length of longest base-paired stretch; (c) total number of base-paired nucleotides. h, Base-pairing frequency along the piRNA length for yw embryo libraries (blue) and their negative control (red). i, Net base-pairing frequency along the piRNA length (red) and net density of base paired nucleotides (grey) in mRNAs from chimaeric CLIP tags from tud embryo libraries. All error bars denote s.d.; n = 3.

Extended Data Figure 8 Non-chimaeric Aub CLIP tag (lgCLIP), chimaeric mRNA fragment and RNA-seq read density along the untranslated and coding sequences of mRNAs.

a, Average density of chimaeric mRNA fragments (Aub CLIP, yw 0–2-h embryo) along the three parts of the meta-mRNA. Each mRNA region is divided in 30 bins and the number of the chimaeric mRNA fragments (genomic coordinate of the mRNA fragment midpoint) mapped within each bin is counted. Inset: bar plot showing cumulative density in each mRNA region. b, Average density of the chimaeric mRNA fragments on mRNA regions; mRNAs are separated into three localization groups as indicated: posterior localized (12 categories; Supplementary Table 3), non-posterior and undetermined localization. Inset: bar plot showing cumulative density in each mRNA region. c, As in a for chimaeric mRNA fragments from Aub CLIP libraries, tud embryo (0–2 h). d, As in b for chimaeric mRNA fragments from Aub CLIP libraries, tud embryo (0–2 h). e, As in a for non-chimaeric lgCLIPs from Aub CLIP libraries, yw embryo (0–2 h). f, As in b for non-chimaeric lgCLIPs from Aub CLIP libraries, yw embryo (0–2 h). g, As in a for non-chimaeric lgCLIPs from Aub CLIP libraries, tud embryo (0–2 h). h, As in b for non-chimaeric lgCLIPs from Aub CLIP libraries, tud embryo (0–2 h). i, As in a for RNA-seq reads, yw embryo (0–2 h). j, As in b for RNA-seq reads, yw embryo (0–2 h). k, As in a for RNA-seq reads, tud embryo (0–2 h). l, As in b for RNA-seq reads, tud embryo (0–2 h). Error bars denote s.d.; n = 3.

Extended Data Figure 9 Lengths of posterior localized mRNAs in Drosophila species; characteristics of embryos used in our studies.

a, Box-and-whisker plot of the number of predicted piRNA target sites (per kilobase of mRNA sequence) for every mRNA–piRNA pair, multiplied by the piRNA copy number. Posterior and non-posterior mRNAs are as indicated. Black lines denote the median. This graph indicates that the ‘targeting potential’ (number of predicted complementary sites multiplied by the piRNA copy number) of each piRNA against each mRNA is the same for the two localization categories, suggesting that the piRNA copy number is not a contributing factor for the observed preference of posterior localized mRNAs for piRNA adhesion. b, Box-and-whisker plot of the lengths of D. melanogaster mRNAs (and their 5′ UTR, coding sequences and 3′ UTR parts) that are found in the enriched and protected categories, as defined previously10. Black lines denote the median; white dots denote the mean. n.s., not significant (P > 0.05); **P <0.01; ***P < 0.001; one-sided Wilcoxon rank sum test. c, Box-and-whisker plot of the lengths of the 3′ UTRs of mRNAs from the indicated Drosophila species that are orthologous to the D. melanogaster mRNAs found in the localized and protected categories, as defined previously10. Incomplete annotation did not allow us to perform this analysis for all the species shown in Fig. 4i.White dots denote the mean. P values of the statistical test (one-sided Wilcoxon test) of whether the lengths of the localized versus protected mRNAs are different, are shown for each species. d, e, RNA-seq scatterplots from 0–2-h wild-type (yw) and 0–2-h Aub-null (aub) embryos. Shown in red are posterior localized mRNAs (d) or the top 100 mRNAs identified from Aub CLIP piRNA–mRNA chimaeric reads (e). There is no change in mRNA levels between wild-type and aub mutant 0–2-h embryos. f, g, Hatch rates (f) and fertility of progeny (g) of embryos from indicated genotypes. Note that, unlike Tudor and Csul, the absence of Aub (aubHN2/QC42) leads to complete embryo lethality. h, Gross ovary appearance of wild-type (yw), Tudor mutant (tud[1/Df]) and Csul mutant (csulRM50) adult flies. Note complete absence of germline ovarian tissue in adult flies lacking Tudor or Csul; embryos from these flies develop into agametic adults because PGCs are never specified.

Extended Data Table 1 Overlap of piRNAs from CLIP and immunoprecipitation libraries
Full size table

Supplementary information

Supplementary Information

This file contains Supplementary Text, Supplementary Results, a Supplementary Discussion and additional references. (PDF 1670 kb)

Supplementary Table 1

Normalized (reads per million, rpm) abundances of piRNA (23-29 nt) and lgClip (>35 nt) subpopulations from the indicated libraries mapping on various transposon consensus sequences (acquired from Repeat Masker). (XLSX 157 kb)

Supplementary Table 2

Statistically significant depletion and enrichment in mRNA localization categories19 in Aub CLIP libraries (non-chimeric lgClips) from yw and tud embryos and yw ovaries (average normalized values from three replicate libraries for each genotype were used). The specificity of Aub for binding posterior localized mRNAs is significantly reduced compared to yw, but not completely lost in tud embryos. (XLSX 98 kb)

Supplementary Table 3

Statistically significant depletion and enrichment in mRNA localization categories19 for pairwise comparison between yw and tud embryo (0-2 h) Aub CLIP libraries (non-chimeric lgClips) (two-sided t-test). (XLSX 67 kb)

Supplementary Table 4

Ranked list of highly bound by Aub (non-chimeric lgClips) posterior localized mRNAs (twelve posterior localization categories marked with an asterisk in Supplementary Table 3). “Localized” (enriched) and “protected” (appearing localized in the pole cells only after the degradation of the maternally deposited mRNAs from the somatic part of the early embryo) mRNAs in the posterior and in the germ cells according to Rangan et al10 are also noted. (XLSX 74 kb)

Supplementary Table 5

Number of unmapped and piRNA:mRNA chimeric reads identified in each CLIP library. The number of chimeric mRNA fragments overlapping with non-chimeric reads for every library is also shown. (XLSX 49 kb)

Supplementary Table 6

Enriched and depleted mRNA localization categories for mRNA fragments extracted from chimeric reads from each set of CLIP libraries (yw and tud embryos, yw ovaries), and also for the comparison between yw and tud embryo libraries. (XLSX 85 kb)

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Vourekas, A., Alexiou, P., Vrettos, N. et al. Sequence-dependent but not sequence-specific piRNA adhesion traps mRNAs to the germ plasm. Nature 531, 390–394 (2016). https://doi.org/10.1038/nature17150

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