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piRNA-mediated regulation of transposon alternative splicing in the soma and germ line

Nature volume 552, pages 268–272 (2017)Cite this article

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Abstract

Transposable elements can drive genome evolution, but their enhanced activity is detrimental to the host and therefore must be tightly regulated1. The Piwi-interacting small RNA (piRNA) pathway is vital for the regulation of transposable elements, by inducing transcriptional silencing or post-transcriptional decay of mRNAs2. Here we show that piRNAs and piRNA biogenesis components regulate precursor mRNA splicing of P-transposable element transcripts in vivo, leading to the production of the non-transposase-encoding mature mRNA isoform in Drosophila germ cells. Unexpectedly, we show that the piRNA pathway components do not act to reduce transcript levels of the P-element transposon during P–M hybrid dysgenesis, a syndrome that affects germline development in Drosophila3,4. Instead, splicing regulation is mechanistically achieved together with piRNA-mediated changes to repressive chromatin states, and relies on the function of the Piwi–piRNA complex proteins Asterix (also known as Gtsf1)5,6,7 and Panoramix (Silencio)8,9, as well as Heterochromatin protein 1a (HP1a; encoded by Su(var)205). Furthermore, we show that this machinery, together with the piRNA Flamenco cluster10, not only controls the accumulation of Gypsy retrotransposon transcripts11 but also regulates the splicing of Gypsy mRNAs in cultured ovarian somatic cells, a process required for the production of infectious particles that can lead to heritable transposition events12,13. Our findings identify splicing regulation as a new role and essential function for the Piwi pathway in protecting the genome against transposon mobility, and provide a model system for studying the role of chromatin structure in modulating alternative splicing during development.

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Figure 1: P-element DNA transposon splicing is regulated in germ cells during hybrid dysgenesis.
Figure 2: piRNAs, but not siRNAs, modulate P-element splicing in germ cells.
Figure 3: piRNA-mediated chromatin changes regulate P-element splicing.
Figure 4: piRNA-mediated chromatin targeting machinery modulates splicing of the Gypsy retrotransposon in OSCs.

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Acknowledgements

We thank A. Zamparini and J. Seifert for early discussions and assistance with developmental analysis; L. Barton for assistance with PGC sorting; T. Trcek for advice on RNA FISH; R. Sachidanandam for small RNA analysis assistance; J. Brennecke, P. Andersen and F. Roudier for advice on ChIP; A. Pelisson for early discussions; W. Theurkauf for antibody reagent; Q. Wang for assistance with JUM analysis; P. Macdonald, R. Carthew, E. Lai, P. Zamore, the Vienna Drosophila Resource Center, and the Bloomington Drosophila Stock Center for fly reagents; the NYUMC Genome Technology Center and the NYUMC Cytometry Center, supported by NIH P30CA016087, for sequencing and cell sorting support. C.D.M. was supported by a HHWF fellowship. F.K.T. was supported by EMBO and HFSP fellowships, and is funded by the Wellcome Trust Sir Henry Dale Fellowship (206257/Z/17/Z). R.L. is supported by NIH R37HD41900 and is an HHMI investigator. D.R. is supported by NIH R35-118121-02 and NIH R01-097352.

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Author notes

  1. Felipe Karam Teixeira

    Present address: Department of Genetics, University of Cambridge, Downing Street, Cambridge, CB2 3EH, UK

  2. Colin D. Malone

    Present address: Institute for Genomic Medicine, Columbia University Medical Center, New York, New York, 10032, USA

Authors and Affiliations

  1. Department of Cell Biology, Howard Hughes Medical Institute (HHMI) and Kimmel Center for Biology and Medicine of the Skirball Institute, New York University School of Medicine, New York, 10016, New York, USA

    Felipe Karam Teixeira, Martyna Okuniewska, Colin D. Malone, Rémi-Xavier Coux & Ruth Lehmann

  2. Department of Molecular and Cell Biology, Center for RNA Systems Biology, and California Institute for Quantitative Biosciences, University of California, Berkeley, 94720, California, USA

    Donald C. Rio

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Contributions

F.K.T. and R.L. conceived the idea. F.K.T., R.L. and D.R. designed the experiments. F.K.T. and M.O. performed the experiments, with the help of C.D.M. and R.C. F.K.T., R.L. and D.R. wrote the manuscript with input from all authors.

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Correspondence to Felipe Karam Teixeira or Ruth Lehmann.

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Reviewer Information Nature thanks J. Brennecke, H. Malik, A. Molaro and the other anonymous reviewer(s) for their contribution to the peer review of this work.

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Extended data figures and tables

Extended Data Figure 1 Size distribution of small RNAs matching P-elements in small RNA libraries.

Libraries were obtained from Harwich ovaries (orange) or from 0–2-h-old embryos laid by Harwich females (purple). Analysis was performed on data from ref. 16. siRNA (20–22 nucleotides long) and piRNA (23–29 nucleotides long) populations are indicated.

Extended Data Figure 2 P-element mRNA steady-state levels do not change in piRNA mutants in comparison to respective heterozygous.

a, b, Scatterplots showing the expression of genes (grey dots) and transposons (blue dots), as measured by RNA-seq analysis (expressed in fragments per kilobase per million fragments (FPKM), log10), in aub/+ heterozygous versus aub mutant adult ovaries (a) and piwi/+ heterozygous versus piwi mutant adult ovaries (b) comparisons. P-element expression is shown in green. Transposons with >10-fold change in RNA abundance are outlined in red. Experiments were repeated twice with similar results. c, d, Genome-wide analysis of splicing changes in aub/+ heterozygous versus aub mutant adult ovaries (c) and piwi/+ heterozygous versus piwi mutant adult ovaries (d) comparisons. Quantification of splicing changes was performed using RNA-seq data and the JUM method44,45. Results are expressed as log2 fold changes in splicing (mutant/heterozygous). Grey dots represent individual splice junctions identified, sorted by fold change values. Green dots represent splice junctions with statistically significant changes in heterozygous versus mutant comparisons (adjusted P < 0.05). Fold changes for P-element splice junctions (IVS1, IVS2 and IVS3) are presented in red. Note that approximately 70% of genic splice junctions showing statistically significant changes in mutant comparisons (green dots) are located in the same chromosome as the inducing mutations (second chromosome), and may be due to genetic background differences. e, Density plots for normalized strand-specific mRNA steady-state levels (measured by RNA-seq) over consensus P-element sequence (top diagram) in piwi/+ heterozygous (beige, top plot) and piwi mutant (purple, bottom plot) adult ovaries. The number and position of split-reads (represented by arcs that connect exons) observed for IVS1, IVS2 and IVS3 splicing junctions are shown below each density plot. Experiments were repeated twice with similar results.

Extended Data Figure 3 P-cytotype and piRNA pathway components involved in piRNA biogenesis and targeting regulate the splicing of the IVS3 transgenic reporter in vivo.

a, Ethidium bromide-stained gel displaying RT–PCR reactions with primers flanking the transgenic reporter IVS3 intron. Analysis was performed with adult ovaries of non-dysgenics and dysgenic flies grown at 18 °C, or with adult ovaries of heterozygous and mutants for the piRNA components aub, vas and Panx. Mutant analyses were performed in a Harwich background, at 29 °C. Size scale in base pairs is presented for each gel. Control reactions omitting reverse transcriptase (RT−) are also presented. Diagram of IVS3 transgenic reporter (as in Fig. 3a) and primers (arrows) used in RT–PCR reactions are depicted in the top of the ethidium bromide-stained gel. Experiments were repeated at least twice with similar results. For gel source data, see Supplementary Fig. 1. b, RT–qPCR analysis using adult ovaries of F1 progeny carrying the IVS3 reporter, probing spliced (IVS3 splicing, quantified using primers that specifically anneal to spliced transgenic transcripts) and total (LacZ, quantified using primers that anneal within the LacZ coding sequence) IVS3 reporter transgenic mRNA levels. F1 progeny originated either from reciprocal crosses between Harwich and w1118 strain flies, or in aub/+ heterozygous, aub mutant, vas/+ heterozygous, vas mutant, panx/+ heterozygous, and panx mutant. Results are presented as mean fold changes in the mutants (or in dysgenic) in relation to the respective heterozygous siblings (or non-dysgenic) ± s.d. (n ≥ 2 independent biological replicate experiments).

Extended Data Figure 4 Loss of H3K9me3 in dysgenic progeny is restricted to transcriptionally active P-elements.

a, b, Density plots for normalized H3K9me3 ChIP–seq signals over consensus P-element (a) and Het-A (b) sequences in non-dysgenic (grey) and dysgenic (red) progeny. Experiments were performed with adult ovaries of flies grown at 18 °C. Experiments were repeated twice with similar results. c, d, H3K9me3 ChIP–qPCR analysis on P-element (c) and controls (d) in non-dysgenic (grey) and dysgenic (red) progeny. Bars represent means of H3K9me3 signal, normalized to a control genomic region in the 42AB locus (42AB-3) ± s.d. (n = 2 independent biological replicate experiments). d, und and Dmn are negative controls. e, f, Genome browser view of two of the P-element insertions showing transcriptional activity. Normalized RNA-seq and H3K9me3 ChIP signals are presented in grey and blue, respectively. Grey bar crossing the plots represents P-element insertion site. Chromosome coordinates are at the top. Annotation is at the bottom: purple boxes, coding exons; pink boxes, untranslated regions; purple lines, introns; grey box, P-element insertion. View showing P-element insertion into CG5953 (e) and CR43651 (f) genes. Experiments were repeated twice with similar results. g, h, ChIP–qPCR analysis on sequences flanking transcriptionally active P-element insertions (g) and on the IVS3 transgenic reporter (h) in non-dysgenic (grey) and dysgenic (red) progeny. Bars represent means of H3K9me3 signal, normalized to a control genomic region in the 42AB locus (42AB-3), ± s.d. (n = 2 independent biological replicate experiments). h, Diagram of IVS3 transgenic reporter is presented below the graph. Black rectangles indicate regions probed by ChIP–qPCR. i, j, Heat maps depicting normalized H3K9me3 levels obtained by ChIP–seq analysis within the 6-kb flanking P-element insertions in non-dysgenic (left) and dysgenic (right) progeny. Experiments were repeated twice with similar results. i, 4 P-element insertions with evidence of transcriptional activity. j, 59 P-element insertions with no evidence of transcriptional activity. All experiments were performed with ovaries of adult progeny from non-dysgenic and dysgenic crosses grown at 18 °C.

Extended Data Figure 5 Characterization of euchromatic P-element insertion in Harwich strain.

a, Ethidium bromide-stained gels displaying genomic PCR reactions with 25 primer sets flanking P-element insertions uncovered by DNA-seq analysis. PCR reactions were performed with genomic DNA extract from w1118 and Harwich pools containing 20 adult females each. Size scale in base pairs is presented on the right side of each gel. Amplicon sizes representing absence (no insertion) or presence (truncated or full-length elements) of P-element insertion are indicated on the left of each gel. Targeted insertion and chromosome localization are displayed at the bottom of each gel. Experiments were repeated twice with similar results. For gel source data, see Supplementary Fig. 1. b, Schematic representation of five structurally different elements regrouping 24 P-element insertions characterized by DNA sequencing in the Harwich strain. Elements size, as well as a list of respective insertions is indicated on the right of each diagram. Arrowheads represent terminal inverted repeats; boxes, exons; inverted triangles, introns. Dashed lines represent internal deletions.

Extended Data Figure 6 Analysis of P-element expression and splicing in adult ovaries of non-dysgenic and dysgenic progeny grown at 18 °C.

a, Scatterplot showing the expression of genes (grey dots) and transposons (blue dots), as measured by RNA-seq analysis (log10), in adult ovaries of non-dysgenic versus dysgenic progeny grown at 18 °C. P-element expression is shown in green. Genes containing a P-element insertion in Harwich strain are depicted in purple. b, c, Percentage of splicing for P-element IVS1, IVS2 (b), and IVS3 (c) splicing junctions as determined by RNA-seq analysis in non-dysgenic (green) and dysgenic (red) adult ovaries. Bars represent percentage of splicing, calculated as the number of split-reads for each splicing junction normalized to the total number of reads mapping to the same junction. Data are mean ± s.d. (n = 2 independent biological replicate experiments). d, Ethidium bromide-stained gel displaying RT–PCR reactions with primers flanking the P-element IVS3 intron in adult ovaries of non-dysgenic and dysgenic progeny grown at 18 °C, as well as aub/+ heterozygous and aub mutant grown at 29 °C. Size scale in base pairs is presented. As shown, experiments were repeated three times with similar results. For gel source data, see Supplementary Fig. 1. e, RT–qPCR analysis testing accumulation of IVS3-spliced mRNA on non-dysgenic and dysgenic progeny (ovaries) grown at 18 °C. Results are expressed as mean of percentage of expression relative to controls ± s.d. (n = 3 independent biological replicate experiments). f, Genome-wide analysis of splicing changes in in adult ovaries of non-dysgenic versus dysgenic progeny grown at 18 °C. Quantification of splicing changes was performed using RNA-seq data and the JUM method44,45. Results are expressed as log2 fold changes in splicing (dysgenic/non-dysgenic). Grey dots represent individual splice junctions identified, sorted by fold change values. Green dots represent splice junctions with statistically significant changes in heterozygous versus mutant comparisons (adjusted P < 0.05). Fold changes for P-element splice junctions (IVS1, IVS2 and IVS3) are presented in red. g, Analysis of RNA-seq data obtained from non-dysgenic (green) and dysgenic (red) progeny (adult ovaries) to identify transcriptionally active P-element insertions. Reads partially mapping to the P-element extremities were subsequently mapped to the Drosophila genome. Results are expressed as percentage of reads mapping to a given genomic location in relation to total reads. Data are mean ± s.d. (n = 2 independent biological replicate experiments). All experiments were performed with ovaries of adult progeny from non-dysgenic and dysgenic crosses grown at 18 °C.

Extended Data Figure 7 Analysis of Gypsy splicing in OSCs.

a, Diagram of Gypsy retrotransposon, as in Fig. 4a. b, RNA-seq signal at the Gypsy splicing donor and acceptor sites in representative control (GFP knockdown) and knockdowns of arx, Panx, Su(var)205, mael, H1, SetDB1, Su(var)3-9 and G9a. Data for double knockdowns of SetDB1 + Su(var)3-9, SetDB1 + G9a, and Su(var.)3-9 + G9a are also shown. With the exception of arx, Su(var)205, mael and H17,25, experiments were repeated twice with similar results. c, Percentage of splicing for Gypsy donor and acceptor splicing sites as determined by RNA-seq analysis performed in OSCs double knockdowns of H3K9 methyltransferases. Bars represent the number of split-reads for the env donor and acceptor splicing junctions normalized to the total number of sense Gypsy reads mapping to the same junction. Results are represented as means. Experiments were repeated twice with similar results. d, Coverage of Gypsy consensus sequence by RNA-seq data. Coverage was calculated as: (number of reads matching Gypsy consensus sequence × read length in nucleotides)/length of consensus Gypsy sequence in nucleotides (7,469 nucleotides). Raw datasets are from refs 7, 9 and 25.

Extended Data Figure 8 rhino ChIP–qPCR analysis in adult ovaries of non-dysgenic and dysgenic progeny grown at 18 °C.

rhino ChIP–qPCR analysis on P-element (a), controls (b), sequences flanking transcriptionally active P-insertions in the Harwich strain (c), and transgenic IVS3 splicing reporter (d) in non-dysgenic (grey) and dysgenic (red) progeny. Bars represent means of Rhino signal, normalized to a control genomic region in the 42AB locus (42AB-3), ± s.d. (n = 2 independent biological replicate experiments). b, Dmn, und and flam are negative controls; 42AB is a positive control. d, Diagram of transgenic IVS3 splicing reporter is presented below the graph. Black rectangles indicate regions probed by ChIP–qPCR. All experiments were performed with ovaries of adult progeny from non-dysgenic and dysgenic crosses grown at 18 °C.

Supplementary information

Supplementary Information

This file contains a Supplementary Note and Supplementary Figure 1, the uncropped gels. (PDF 397 kb)

Life Sciences Reporting Summary (PDF 137 kb)

Supplementary Table 1

This table contains euchromatic P-element insertions in Harwich strain, as determined by DNA-seq analysis. (XLSX 60 kb)

Supplementary Table 2

This table contains a description of Harwich-derived stocks. (XLSX 9 kb)

Supplementary Table 3

This table contains a list of crosses. (XLSX 10 kb)

Supplementary Table 4

This table contains a list of RNA FISH probes. (XLSX 49 kb)

Supplementary Table 5

This table contains a list of primers. (XLSX 45 kb)

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Teixeira, F., Okuniewska, M., Malone, C. et al. piRNA-mediated regulation of transposon alternative splicing in the soma and germ line. Nature 552, 268–272 (2017). https://doi.org/10.1038/nature25018

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