The piRNA Response to Retroviral Invasion of the Koala Genome

Transposons are ubiquitous mobile elements with the potential to trigger genome instability and mutations linked to diseases1,2. Antisense piRNAs guide an adaptive genome immune system that silences established transposons during germline development3, but how the germline responds to new genome invaders is not understood. The KoRV retrovirus infects somatic and germline cells and is sweeping through wild koala populations by a combination of horizontal and vertical transfers, providing a unique opportunity to directly analyze the germline response to retroviral invasions of a mammalian genome4,5. We analyzed genome organization and long RNA and short RNA transcriptomes in testis, liver, and brain from two wild koalas infected with KoRV, while integrating our results with earlier genomic data. Consistent with data from other mammals6,7, koala piRNAs were detected in testis and mapped to both isolated transposon insertions and genic and intergenic piRNA clusters. Established transposon subfamilies produced roughly equal levels of antisense piRNAs, which are the effectors of trans-silencing, and sense piRNAs, which drive ping-pong amplification of these effectors8,9. KoRV piRNAs, in striking contrast, were strongly sense biased in both animals analyzed. These two koalas each carried 60 germline KoRV-A insertions, but only 14 of the insertions were shared, and none of the insertions mapped to piRNA clusters. The sense piRNAs thus appear to be produced by direct processing of the transcripts from isolated proviral insertions. A typical gammaretrovirus, KoRV produces spliced Env mRNAs and unspliced transcripts encoding Gag, Pol, and the viral genome. KoRV Env mRNAs were 5-fold more abundant than the unspliced pre-mRNAs, but 92% of piRNAs were derived from the unspliced pre-mRNAs. We show that this biased piRNA production from unspliced retrotransposon transcripts is conserved from flies to mice. Retroviruses must bypass splicing to replicate; thus, we propose that failed splicing produces a “molecular pattern” on transcripts from retroviral invaders that is recognized by an innate genome immune system, which silences transposons in cis by processing their transcripts into piRNAs. This innate immune response defends the germline until antisense piRNA production—from clusters or isolated insertions—is established to provide sequence-specific adaptive immunity and memory of the genome invader.

[1]  Peter R Andersen,et al.  A Heterochromatin-Specific RNA Export Pathway Facilitates piRNA Production , 2019, Cell.

[2]  W. Theurkauf,et al.  Rapid evolution and conserved function of the piRNA pathway , 2018, Royal Society Open Biology.

[3]  Z. Weng,et al.  Co-dependent Assembly of Drosophila piRNA Precursor Complexes and piRNA Cluster Heterochromatin. , 2018, Cell reports.

[4]  Graham J. Etherington,et al.  Adaptation and conservation insights from the koala genome , 2018, Nature Genetics.

[5]  J. Luban,et al.  Primate immunodeficiency virus Vpx and Vpr counteract transcriptional repression of proviruses by the HUSH complex , 2018, bioRxiv.

[6]  Zhiping Weng,et al.  The genome of the Hi5 germ cell line from Trichoplusia ni, an agricultural pest and novel model for small RNA biology , 2018, eLife.

[7]  D. Odom,et al.  The emergence of piRNAs against transposon invasion to preserve mammalian genome integrity , 2017, Nature Communications.

[8]  Z. Weng,et al.  Adaptive Evolution Leads to Cross-Species Incompatibility in the piRNA Transposon Silencing Machinery. , 2017, Developmental cell.

[9]  A. Hayward,et al.  Origin of the retroviruses: when, where, and how? , 2017, Current opinion in virology.

[10]  S. Waddell,et al.  Resolving the prevalence of somatic transposition in Drosophila , 2017, eLife.

[11]  E. Holmes,et al.  Phylogenetic Diversity of Koala Retrovirus within a Wild Koala Population , 2016, Journal of Virology.

[12]  A. Aravin,et al.  Splicing-independent loading of TREX on nascent RNA is required for efficient expression of dual-strand piRNA clusters in Drosophila , 2016, Genes & development.

[13]  A. Roca,et al.  Comprehensive profiling of retroviral integration sites using target enrichment methods from historical koala samples without an assembled reference genome , 2016, PeerJ.

[14]  F. He,et al.  Nonsense-Mediated mRNA Decay: Degradation of Defective Transcripts Is Only Part of the Story. , 2015, Annual review of genetics.

[15]  W. Johnson,et al.  Endogenous Retroviruses in the Genomics Era. , 2015, Annual review of virology.

[16]  M. Mougel,et al.  Insights into the nuclear export of murine leukemia virus intron-containing RNA , 2015, RNA biology.

[17]  Oliver H. Tam,et al.  RNF17 blocks promiscuous activity of PIWI proteins in mouse testes , 2015, Genes & development.

[18]  Zhiping Weng,et al.  piRNA-guided transposon cleavage initiates Zucchini-dependent, phased piRNA production , 2015, Science.

[19]  Julius Brennecke,et al.  piRNA-guided slicing specifies transcripts for Zucchini-dependent, phased piRNA biogenesis , 2015, Science.

[20]  Sky W. Brubaker,et al.  Innate immune pattern recognition: a cell biological perspective. , 2015, Annual review of immunology.

[21]  R. Ketting,et al.  Piwi proteins and piRNAs in mammalian oocytes and early embryos. , 2015, Cell reports.

[22]  P. Alexiou,et al.  The RNA helicase MOV10L1 binds piRNA precursors to initiate piRNA processing , 2015, Genes & development.

[23]  Steven L Salzberg,et al.  HISAT: a fast spliced aligner with low memory requirements , 2015, Nature Methods.

[24]  A. Roca,et al.  Proliferation of endogenous retroviruses in the early stages of a host germ line invasion. , 2015, Molecular biology and evolution.

[25]  E. Miska,et al.  piRNAs: from biogenesis to function , 2014, Development.

[26]  M. Wilkins,et al.  A transcriptome resource for the koala (Phascolarctos cinereus): insights into koala retrovirus transcription and sequence diversity , 2014, BMC Genomics.

[27]  Paul Theodor Pyl,et al.  HTSeq—a Python framework to work with high-throughput sequencing data , 2014, bioRxiv.

[28]  R. Sachidanandam,et al.  Transgenerationally inherited piRNAs trigger piRNA biogenesis by changing the chromatin of piRNA clusters and inducing precursor processing , 2014, Genes & development.

[29]  Fabio Mohn,et al.  The Rhino-Deadlock-Cutoff Complex Licenses Noncanonical Transcription of Dual-Strand piRNA Clusters in Drosophila , 2014, Cell.

[30]  Z. Weng,et al.  The HP1 Homolog Rhino Anchors a Nuclear Complex that Suppresses piRNA Precursor Splicing , 2014, Cell.

[31]  Zhiping Weng,et al.  TEMP: a computational method for analyzing transposable element polymorphism in populations , 2014, Nucleic acids research.

[32]  Y. Ikeda,et al.  Murine Leukemia Virus Uses TREX Components for Efficient Nuclear Export of Unspliced Viral Transcripts , 2014, Viruses.

[33]  Q. Fu,et al.  Mammalian piRNAs , 2014, Spermatogenesis.

[34]  Matthias Zytnicki,et al.  Distribution, evolution, and diversity of retrotransposons at the flamenco locus reflect the regulatory properties of piRNA clusters , 2013, Proceedings of the National Academy of Sciences.

[35]  P. Young,et al.  Koala retroviruses: characterization and impact on the life of koalas , 2013, Retrovirology.

[36]  T. Mikkelsen,et al.  Cellular source and mechanisms of high transcriptome complexity in the mammalian testis. , 2013, Cell reports.

[37]  Aviv Regev,et al.  Comprehensive comparative analysis of RNA sequencing methods for degraded or low input samples , 2013, Nature Methods.

[38]  Zhiping Weng,et al.  An ancient transcription factor initiates the burst of piRNA production during early meiosis in mouse testes. , 2013, Molecular cell.

[39]  D. Bartel,et al.  Stalled Spliceosomes Are a Signal for RNAi-Mediated Genome Defense , 2013, Cell.

[40]  S. Ho,et al.  One Hundred Twenty Years of Koala Retrovirus Evolution Determined from Museum Skins , 2012, Molecular biology and evolution.

[41]  Thomas R. Gingeras,et al.  STAR: ultrafast universal RNA-seq aligner , 2013, Bioinform..

[42]  P. Khaitovich,et al.  Birth and expression evolution of mammalian microRNA genes , 2013, Genome research.

[43]  Z. Weng,et al.  Strand-specific libraries for high throughput RNA sequencing (RNA-Seq) prepared without poly(A) selection , 2012, Silence.

[44]  Harrison W. Gabel,et al.  Small RNA pathway genes identified by patterns of phylogenetic conservation and divergence , 2012, Nature.

[45]  C. Burge,et al.  Evolutionary Dynamics of Gene and Isoform Regulation in Mammalian Tissues , 2012, Science.

[46]  C. Brun,et al.  piRNA-mediated transgenerational inheritance of an acquired trait , 2012, Genome research.

[47]  Kunbin Qu,et al.  Selective Depletion of rRNA Enables Whole Transcriptome Profiling of Archival Fixed Tissue , 2012, PloS one.

[48]  Steven L Salzberg,et al.  Fast gapped-read alignment with Bowtie 2 , 2012, Nature Methods.

[49]  Sebastian D. Mackowiak,et al.  miRDeep2 accurately identifies known and hundreds of novel microRNA genes in seven animal clades , 2011, Nucleic acids research.

[50]  Zhiping Weng,et al.  Adaptation to P Element Transposon Invasion in Drosophila melanogaster , 2011, Cell.

[51]  W. Paul,et al.  Bridging Innate and Adaptive Immunity , 2011, Cell.

[52]  Marcel Martin Cutadapt removes adapter sequences from high-throughput sequencing reads , 2011 .

[53]  A. Aravin,et al.  PIWI-interacting small RNAs: the vanguard of genome defence , 2011, Nature Reviews Molecular Cell Biology.

[54]  B. Langmead,et al.  Aligning Short Sequencing Reads with Bowtie , 2010, Current protocols in bioinformatics.

[55]  Robert C. Edgar,et al.  BIOINFORMATICS APPLICATIONS NOTE , 2001 .

[56]  Andrew F. Neuwald,et al.  Natural Mutagenesis of Human Genomes by Endogenous Retrotransposons , 2010, Cell.

[57]  Aaron R. Quinlan,et al.  Bioinformatics Applications Note Genome Analysis Bedtools: a Flexible Suite of Utilities for Comparing Genomic Features , 2022 .

[58]  F. Bonilla,et al.  Adaptive immunity. , 2010, The Journal of allergy and clinical immunology.

[59]  S. Kurtz,et al.  Fine-grained annotation and classification of de novo predicted LTR retrotransposons , 2009, Nucleic acids research.

[60]  Z. Weng,et al.  The Drosophila HP1 Homolog Rhino Is Required for Transposon Silencing and piRNA Production by Dual-Strand Clusters , 2009, Cell.

[61]  Gonçalo R. Abecasis,et al.  The Sequence Alignment/Map format and SAMtools , 2009, Bioinform..

[62]  Richard Durbin,et al.  Sequence analysis Fast and accurate short read alignment with Burrows – Wheeler transform , 2009 .

[63]  Z. Weng,et al.  Collapse of Germline piRNAs in the Absence of Argonaute3 Reveals Somatic piRNAs in Flies , 2009, Cell.

[64]  R. Sachidanandam,et al.  An Epigenetic Role for Maternally Inherited piRNAs in Transposon Silencing , 2008, Science.

[65]  C. Brun,et al.  piRNA-mediated nuclear accumulation of retrotransposon transcripts in the Drosophila female germline , 2008, Proceedings of the National Academy of Sciences.

[66]  Ravi Sachidanandam,et al.  A piRNA pathway primed by individual transposons is linked to de novo DNA methylation in mice. , 2008, Molecular cell.

[67]  P. Deininger,et al.  Mammalian non-LTR retrotransposons: for better or worse, in sickness and in health. , 2008, Genome research.

[68]  Stefan Kurtz,et al.  LTRharvest, an efficient and flexible software for de novo detection of LTR retrotransposons , 2008, BMC Bioinformatics.

[69]  G. Hannon,et al.  The Piwi-piRNA Pathway Provides an Adaptive Defense in the Transposon Arms Race , 2007, Science.

[70]  Peng Wang,et al.  The Drosophila RNA Methyltransferase, DmHen1, Modifies Germline piRNAs and Single-Stranded siRNAs in RISC , 2007, Current Biology.

[71]  Kuniaki Saito,et al.  Pimet, the Drosophila homolog of HEN1, mediates 2'-O-methylation of Piwi- interacting RNAs at their 3' ends. , 2007, Genes & development.

[72]  Ravi Sachidanandam,et al.  Developmentally Regulated piRNA Clusters Implicate MILI in Transposon Control , 2007, Science.

[73]  Peter F. Hallin,et al.  RNAmmer: consistent and rapid annotation of ribosomal RNA genes , 2007, Nucleic acids research.

[74]  Zissimos Mourelatos,et al.  Mouse Piwi-interacting RNAs are 2′-O-methylated at their 3′ termini , 2007, Nature Structural &Molecular Biology.

[75]  A. Pélisson,et al.  The flamenco Locus Controls the gypsy and ZAM Retroviruses and Is Required for Drosophila Oogenesis , 2007, Genetics.

[76]  Manolis Kellis,et al.  Discrete Small RNA-Generating Loci as Master Regulators of Transposon Activity in Drosophila , 2007, Cell.

[77]  Kuniaki Saito,et al.  A Slicer-Mediated Mechanism for Repeat-Associated siRNA 5' End Formation in Drosophila , 2007, Science.

[78]  Michael Ashburner,et al.  Recurrent insertion and duplication generate networks of transposable element sequences in the Drosophila melanogaster genome , 2006, Genome Biology.

[79]  Kuniaki Saito,et al.  Specific association of Piwi with rasiRNAs derived from retrotransposon and heterochromatic regions in the Drosophila genome. , 2006, Genes & development.

[80]  J. Meers,et al.  Retroviral invasion of the koala genome , 2006, Nature.

[81]  N. Bannert,et al.  Transspecies Transmission of the Endogenous Koala Retrovirus , 2006, Journal of Virology.

[82]  G. Kao,et al.  Gamma radiation increases endonuclease-dependent L1 retrotransposition in a cultured cell assay , 2006, Nucleic acids research.

[83]  B. Finlay,et al.  Anti-Immunology: Evasion of the Host Immune System by Bacterial and Viral Pathogens , 2006, Cell.

[84]  A. Pélisson,et al.  Evidence for a piwi-Dependent RNA Silencing of the gypsy Endogenous Retrovirus by the Drosophila melanogaster flamenco Gene , 2004, Genetics.

[85]  K. McEntee,et al.  DNA damage activates transcription and transposition of yeast Ty retrotransposons , 1989, Molecular and General Genetics MGG.

[86]  Peter Libby,et al.  Innate and Adaptive Immunity in the Pathogenesis of Atherosclerosis , 2002, Circulation research.

[87]  L. Bromham,et al.  The Nucleotide Sequence of Koala (Phascolarctos cinereus) Retrovirus: a Novel Type C Endogenous Virus Related to Gibbon Ape Leukemia Virus , 2000, Journal of Virology.

[88]  A. Bucheton,et al.  Flamenco, a gene controlling the gypsy retrovirus of Drosophila melanogaster. , 1995, Genetics.

[89]  D. Rio Regulation of Drosophila P element transposition. , 1991, Trends in genetics : TIG.

[90]  B. Mcclintock,et al.  The significance of responses of the genome to challenge. , 1984, Science.

[91]  C. Kozak,et al.  Germ-line reinsertions of AKR murine leukemia virus genomes in Akv-1 congenic mice. , 1980, Proceedings of the National Academy of Sciences of the United States of America.

[92]  J. Hartley,et al.  Genetic Mapping of a Murine Leukemia Virus-Inducing Locus of AKR Mice , 1972, Science.