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A satellite repeat-derived piRNA controls embryonic development of Aedes

Abstract

Tandem repeat elements such as the diverse class of satellite repeats occupy large parts of eukaryotic chromosomes, mostly at centromeric, pericentromeric, telomeric and subtelomeric regions1. However, some elements are located in euchromatic regions throughout the genome and have been hypothesized to regulate gene expression in cis by modulating local chromatin structure, or in trans via transcripts derived from the repeats2,3,4. Here we show that a satellite repeat in the mosquito Aedes aegypti promotes sequence-specific gene silencing via the expression of two PIWI-interacting RNAs (piRNAs). Whereas satellite repeats and piRNA sequences generally evolve extremely quickly5,6,7, this locus was conserved for approximately 200 million years, suggesting that it has a central function in mosquito biology. piRNA production commenced shortly after egg laying, and inactivation of the more abundant piRNA resulted in failure to degrade maternally deposited transcripts in the zygote and developmental arrest. Our results reveal a mechanism by which satellite repeats regulate global gene expression in trans via piRNA-mediated gene silencing that is essential for embryonic development.

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Fig. 1: An evolutionarily conserved satellite repeat produces piRNAs that associate with Piwi4.
Fig. 2: tapiR1 silences target RNAs in trans through seed-mediated base pairing.
Fig. 3: tapiR1 promotes turnover of maternal transcripts and is essential for embryonic development.

Data availability

Raw sequence data have been deposited in the NCBI Sequence Read Archive under BioProject numbers PRJNA482553 and PRJNA594491.

Code availability

The source code is available at https://github.com/RebeccaHalbach/Halbach_tapiR_2020.git.

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Acknowledgements

We thank past and current members of the laboratory for discussions; A. B. Crist and A. Baidaliuk for their help with mosquito rearing and embryo injections; C. Bourgouin and N. Puchot for assistance with the microinjection apparatus; B. Dutilh for discussions about analyses of target-site enrichment; G. -J. van Gemert and M. Kristan for providing mosquitoes; and T. Möhlmann for collecting wild-caught mosquito samples. The following reagent was provided by the NIH/NIAID Filariasis Research Reagent Resource Center for distribution by BEI Resources, NIAID, NIH: Ae. aegypti, strain black eye Liverpool, eggs, NR-48921. Sequencing was performed by the GenomEast platform, a member of the France Génomique consortium (ANR-10-INBS-0009). This work is supported by a Consolidator Grant from the European Research Council under the European Union’s Seventh Framework Programme (grant number ERC CoG 615680) and a VICI grant from the Netherlands Organization for Scientific Research (grant number 016.VICI.170.090). A stay of R.H. at Pasteur Institute, Paris, France was supported by ERASMUS+.

Author information

Authors and Affiliations

Authors

Contributions

R.H., P.M. and R.P.v.R designed the experiments and analysed the data. R.H. performed the computational analyses and most of the experiments, except for PIWI immunoprecipitations for small RNA-seq (J.J. and E.T.), design and validation of PIWI antibodies (B.P.) and tissue isolations and blood feeding experiment (C.B.F.V. and C.J.K.). C.B.F.V. and C.J.K. provided wild-caught mosquito samples. I.R. assisted with the experiments, and S.H.M. and L.L. helped with optimizing embryo injections. R.H. and R.P.v.R. wrote the paper. All authors read and contributed to the manuscript.

Corresponding author

Correspondence to Ronald P. van Rij.

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Competing interests

The authors declare no competing interests.

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Peer review information Nature thanks Francis Jiggins, Susumu Katsuma 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 Fig. 1 Expression of piRNAs from a satellite repeat locus.

a, Fraction of siRNAs and piRNAs mapping on genomic features in adult Ae. aegypti female ovaries (germline) or carcasses (soma). Small RNAs that overlapped multiple features were assigned to only one category (Methods). The leftmost bar depicts the fraction of each feature category in the genome. b, Read length distribution of tapiR1 and 2 in Aag2 cells, and adult germline and somatic tissues (β-eliminated, or untreated).

Extended Data Fig. 2 tapiR1 and 2 are expressed in Ae. aegypti mosquitoes and associate with Piwi4.

a, d, e, Northern blots of tapiR1 and tapiR2 in different tissues of adult mosquitoes (a), upon dsRNA-mediated knockdown of individual PIWI genes (d) and upon knockdown of miRNA-and siRNA-pathway genes (e), or a control dsRNA treatment (dsFLuc and dsRLuc) in Aag2 cells. U6 snRNA or ethidium-bromide-stained rRNA serve as loading controls. b, Western blot analysis of the indicated PIWI proteins before (input) and after immunoprecipitation (IP) used for the small RNA northern blot in c. An immunoprecipitation with empty beads serves as negative control. Tubulin was used to control for nonspecific binding. c, Immunoprecipitation of the indicated PIWI proteins from Aag2 cells followed by northern blot analyses for tapiR1 and tapiR2.

Extended Data Fig. 3 Expression of tapiR1 is conserved in culicine mosquitoes and is independent of AAEL017385.

a, Schematic representation of the tapiR satellite repeat locus in Ae. aegypti, Ae. albopictus and Culex quinquefasciatus. Numbers indicate repeat lengths and, for Culex quinquefasciatus, lengths of deviating repeat monomers. bd, Sequence logos constructed from all individual tapiR repeat units in Ae. aegypti (b), Ae. albopictus (c) or Culex quinquefasciatus (d). Gaps in the sequence logos mainly arise owing to size heterogeneity in few repeat monomers. e, Evolutionary relationships of dipterous genera based on ref. 10. Bar lengths are arbitrary and do not reflect evolutionary distances. f, Northern blot of tapiR1 in Aag2 cells treated with dsRNA targeting different transcripts of AAEL017385 (indicated in g) or, as control, firefly luciferase. Ethidium bromide stained rRNA serves as loading control. g, Top, Schematic of AAEL017385 and the tapiR satellite repeat locus. The primer used for 3′ RACE, and positions targeted by dsRNA in f are indicated with an arrow and wavy lines, respectively. Bottom, 3′ RACE analysis of AAEL017385 transcripts. Indicated are sequences from the current AaegL5 genome annotation and RACE PCR products. The sequences of the 5′ terminal part of tapiR1 and tapiR2 repeats are highlighted with colours. h, Northern blot of a potential tapiR1 or tapiR2 precursor transcript. i, RNA-seq read coverage of the tapiR repeat locus and AAEL017385 (top) and sashimi plot indicating spliced reads (bottom).

Extended Data Fig. 4 Antisense oligonucleotides relieve tapiR1-mediated silencing.

a, Luciferase assay of reporters with a fully complementary target site for tapiR1 (left) or tapiR2 (right) in the 3′ UTR. Aag2 cells were co-transfected with the reporter and increasing amounts of fully 2′O-methylated antisense tapiR RNA oligonucleotides, or a control antisense oligonucleotide. Firefly luciferase activity was normalized to the activity of a co-transfected Renilla luciferase reporter. Indicated are mean, s.d. and individual measurements from a representative experiment measured in triplicate wells. b, Northern blot of tapiR1, tapiR2 and three different miRNAs in Aag2 cells upon treatment with the indicated concentrations of tapiR1, tapiR2 or control antisense oligonucleotides. Ethidium-bromide-stained rRNA serves as loading control.

Extended Data Fig. 5 Renilla luciferase contains a functional tapiR1 target site.

a, Schematic of the different reporter constructs used in this study. pMT, metallothionein promoter; RNAPIII, RNA polymerase III reporter. b, Representative northern blot (left) and quantification (right) of RNAPIII reporters carrying the indicated tapiR1 target sites. Values are normalized to a non-targeted transfection control. Mean, s.d. and individual measures of three independent experiments (indicated with colours), quantified in triplicate, are shown. The panels are split to reflect that samples were loaded at different locations of the same gel. c, Schematic of predicted tapiR1 target sites and minimum free energy of the indicated structures in the coding sequences of Renilla luciferase (RLuc) or firefly luciferase (FLuc). Numbers indicate the position of the targets relative to the first nucleotide in the ORFs. d, Luciferase assay of Aag2 cells transfected with reporters carrying either a scrambled (scr) site or the predicted target site from firefly luciferase (left) or Renilla luciferase (right) from c in the 3′UTR of FLuc. e, Luciferase activity of FLuc or RLuc constructs with synonymous mutations introduced into the predicted tapiR1 target site (ΔtapiR1 site), and the parental clones. f, Luciferase assay of reporters carrying tapiR1 target sites or control sequences in the 3′UTR of either the parental firefly luciferase or the ΔtapiR1 firefly luciferase version. g, Reporter assay with luciferase carrying tapiR1 target sites with single mismatches in the 3′UTR as used in Extended Data Fig. 6b, using RLuc with a mutated tapiR1 target site (ΔtapiR1 site) for normalization. Left, magnified version of the graph on the right. Shown are mean, s.d. and individual measurements from representative experiments performed with at least two different clones per construct, and each measured in triplicate wells.

Extended Data Fig. 6 tapiR1 uses a G:U wobble sensitive seed sequence for target recognition.

a, Schematic of the reporter constructs used in b and Fig. 2. Numbers indicate the position of the mismatch relative to the 5′ end of the piRNA. b, Luciferase activity of reporters carrying a tapiR1 target site with single mismatches. c, Luciferase activity of reporters with the tapiR1 target site from RLuc and indicated mismatches in the 3′UTR of FLuc (left). Right, predicted tapiR1–RLuc  target RNA duplexes. d, Luciferase activity of tapiR1 reporters carrying mismatches or G:U wobble base pairs at the indicated positions. Firefly luciferase activity was normalized to the activity of a co-transfected Renilla luciferase reporter to control for differences in transfection efficiencies. Data represent mean, s.d. and individual measurements of representative experiments with two independent clones per construct and measured in triplicate wells.

Extended Data Fig. 7 Validation of tapiR1 target genes.

a, Predicted structures and minimum free energy of tapiR1–target RNA duplexes analysed in b. Left column, functional target sites resulting in silencing of luciferase reporters; right column, nonfunctional sites. b, Luciferase assay of reporters carrying the predicted target sites from a in the 3′UTR of firefly luciferase. Firefly luciferase activity was normalized to the activity of a co-transfected Renilla luciferase reporter to control for differences in transfection efficiencies. Indicated are mean, s.d. and individual measurements from representative experiments performed with one to three independent clones per construct and measured in triplicate wells. c, AAEL001741, AAEL017422 and AAEL000453 were annotated in the previous AaegL3 gene set, but not in the current AaegL5 gene set. Read coverage in Aag2 cells treated with tapiR1 or control antisense oligonucleotides at these genomic regions suggests that these regions are actively transcribed but repressed by tapiR1. Red boxes indicate the positions of tapiR1 target sites.

Extended Data Fig. 8 tapiR1 and Piwi4 silence gene expression in Aag2 cells.

a, mRNA expression of transposable elements in Aag2 cells treated with a tapiR1-specific antisense oligonucleotide or control antisense oligonucleotide. Depicted are the means of three biological replicates. A pseudocount of one was added to all values to plot values of zero. Diagonal lines represent a twofold change. Significance was tested at an FDR of 0.01 and a log2-transformed fold change of 0.5. b, Luciferase assay of reporters containing different tapiR1 target sites (from Extended Data Fig. 7a) in the 3′UTR of firefly luciferase. Firefly luciferase activity was normalized to the activity of a co-transfected Renilla luciferase reporter to control for differences in transfection efficiencies. Data represent mean, s.d. and individual measurements of representative experiments measured in triplicate wells. c, d, RT–qPCR of tapiR1 target genes upon dsRNA-mediated knockdown of FLuc (control), Piwi4 or Ago1 in Aag2 cells (c), or after treatment with different concentrations of control, tapiR1 or tapiR2 antisense oligonucleotides (d). Depicted are mean, s.d. and individual measurements of a representative experiment as measured from triplicate wells (c), or from duplicate wells (d). Even skipped (eve) does not contain a tapiR1 target site and serves as control. e, Violin plot of log2-transformed fold changes in mRNA expression of all genes upon treatment with tapiR1 or control antisense oligonucleotides in Aag2 cells (left) and mosquito embryos (right), either with or without predicted tapiR1 target site. f, log2-transformed fold changes in RNA expression of genes upon treatment with tapiR1 or control antisense oligonucleotides in Aag2 cells (left) and mosquito embryos (right) plotted against the minimum free energy of predicted tapiR1–target RNA duplexes. Blue dots indicate target sites that were confirmed to be functional in luciferase reporter assays, and red dots indicate target sites that were not functional (Extended Data Fig. 7b).

Extended Data Fig. 9 tapiR1 regulates gene expression in mosquito embryos.

a, Western blot analysis of phosphorylated RNA polymerase II (serine 2 of the C-terminal domain repeats, middle) and tubulin (bottom panel) in embryos at the indicated time points after egg laying, and corresponding stem-loop RT–qPCR of tapiR1 measured in technical duplicates (top). b, c, Northern blot of tapiR1 and 2 in developmental stages of Ae. aegypti mosquitoes (b), or at different time points after blood feeding (c). U6 snRNA (b) or ethidium bromide-stained rRNA (c) were analysed to verify equal loading. d, e, Piwi4 RNA-seq read counts in the indicated adult tissues (d) or developmental stages (e). Libraries used for these analyses are listed in Supplementary Table 6. f, log2-transformed mRNA expression of transposable elements in embryos injected with tapiR1 or control antisense oligonucleotides. Mean counts of five biological replicates are shown. Significance was tested at an FDR of 0.01 and a log2-transformed fold change of 0.5. Diagonal lines indicate a twofold change. g, h, RT–qPCR of the indicated tapiR1 target genes 9 h after injection of tapiR1 or control antisense oligonucleotides (g), or dsRNA-mediated knockdown of FLuc (control), Piwi4 or Ago1 (h) 21 h after injection in embryos. Mean, s.d. and individual measurements of a representative experiment are presented. Even skipped (eve) is not a tapiR1 target gene and serves as negative control. i, Overlap of upregulated tapiR1 target genes (log2-transformed fold change ≥ 1, with a predicted target site with mfe ≤ −24 kcal/mol) in Aag2 cells and Ae. aegypti embryos.

Extended Data Fig. 10 tapiR1 regulates gene expression at a post-transcriptional level.

a, Schematic of different modes of silencing of the three small RNA silencing pathways in Drosophila. b, Top, sequences of tapiR1, target gene AAEL026349, an siRNA targeting the gene at the same position, and Sanger sequencing results of 5′ RACE of Aag2 cells treated with tapiR1 antisense oligonucleotide and siRNAs. The tapiR1 target site is indicated in blue, RACE-sequencing adaptor in yellow and gene sequence in dark grey. The predicted slice site between nucleotides 10 and 11 is marked with a red vertical line. Bottom, summary of the results from 5′ RACE in the indicated conditions. Numbers refer to the number of sequenced clones with the 5′ RACE adaptor ligated to the predicted slice site and the total number of sequenced clones is shown between brackets. c, Small RNA coverage in Aag2 cells (not normalized) and individual reads (direction of the arrow indicates the strand) on tapiR1 target genes. Red boxes indicate positions of tapiR1 target sites on the mRNA. d, Schematic (top) and luciferase expression (bottom) of IRES-containing reporter constructs. Depicted are mean, s.d. and individual measurements of a representative experiment performed in triplicate wells with two different reporter clones. Bottom right, firefly luciferase (FLuc) activity normalized to Renilla luciferase (RLuc); raw luciferase counts from the same experiment are shown in the left (FLuc) and middle (RLuc). e, Luciferase activity of a reporter harbouring the tapiR1 target site of AAEL001555 in the 3′UTR of FLuc upon dsRNA-mediated knockdown of the indicated genes. Symbols are colour-coded according to the indicated RNA decay pathways. FLuc expression was normalized to RLuc expression to control for differences in transfection efficiencies and expressed relative to non-targeting control dsRNA (Sindbis virus dsRNA). Depicted are mean and standard deviation of one experiment performed in triplicates. Horizontal lines indicate a fold change of 1 and 1.5. f, AAEL008511 target gene expression as measured by RT–qPCR upon knockdown of the indicated genes. Primer sets located 5′ (upstream) and 3′ (downstream) to the tapiR1 target site were used for PCR. Mean, s.d. and individual measurements of one out of three experiments performed in triplicate are shown. The other two experiments are presented in Supplementary Fig. 2f, g. The horizontal line indicates a twofold change. g, Schematic illustration of the two PAT assays and expected results of genes with increasing poly(A) tail lengths. LM-PAT, ligation mediated-PAT; RACE-PAT, rapid amplification of cDNA ends-PAT. hj, Electrophoretic analysis with ethidium-bromide-stained agarose gels of a LM-PAT assay (h) and RACE-PAT assay (i) of different tapiR1 target genes upon treatment with two concentrations of tapiR1 or control antisense oligonucleotides. As positive control, poly(A)-tail length was measured from SINV RNA in vitro transcribed from a plasmid (IVT), or from infected Aag2 cells, in which the poly(A) tail is elongated during viral replication56 (j). White asteriks indicate primer dimers.

Supplementary information

Supplementary Information

This file contains Supplementary Discussions 1-2, Supplementary References, Supplementary Fig. 1 (the uncropped northern blots and Western blot corresponding to the indicated figures) and Supplementary Fig. 2.

Reporting Summary

Supplementary Table 1

Differentially expressed genes upon tapiR1 AO treatment in Aag2 cells (baseMean: average of normalized count values; lfcSE: log fold change standard error; stat: Wald statistic; pval: p-value; padj: adjusted p-value).

Supplementary Table 2

Differentially expressed transposable elements upon tapiR1 AO treatment in Aag2 cells.

Supplementary Table 3

Differentially expressed genes upon tapiR1 AO treatment in embryos.

Supplementary Table 4

Differentially expressed transposable elements upon tapiR1 AO treatment in embryos.

Supplementary Table 5

Oligonucleotide sequences used in this study.

Supplementary Table 6

RNA sequencing datasets used in this study.

Supplementary Table 7

Detailed information for each figure panel.

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Halbach, R., Miesen, P., Joosten, J. et al. A satellite repeat-derived piRNA controls embryonic development of Aedes. Nature 580, 274–277 (2020). https://doi.org/10.1038/s41586-020-2159-2

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