Co-dependent Assembly of Drosophila piRNA Precursor Complexes and piRNA Cluster Heterochromatin

Zhang, Gen; Tu, Shikui; Yu, Tianxiong; Zhang, Xiao‐Ou; Parhad, Swapnil S.; Weng, Zhiping; Theurkauf, William E.

doi:10.1016/j.celrep.2018.08.081

Cited by 33 publications

(41 citation statements)

References 55 publications

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“…It is plausible that Nxt1 is required for export by Nxf3, or it might function in co-transcriptional recruitment to cargo RNAs. In this regard, it is striking that Nxt1, and also UAP56 and THO complex components are concentrated at piRNA clusters (Zhang et al 2012;Zhang et al 2018), even though these proteins are reportedly essential for general mRNA export.…”

Section: Discussionmentioning

confidence: 99%

“…Thus, this Rhi-anchored complex generates transcripts that lack signatures of canonical mRNAs. UAP56, a DEAD-box helicase also involved in canonical mRNA splicing and export, was shown to associate with piRNA precursor transcripts (Zhang et al 2012;Zhang et al 2018). Co-localization of UAP56 with the sites of dual-strand cluster transcription and a close association with the perinuclear DEAD-box helicase Vasa (Vas) suggested that this protein is involved in coupling precursor synthesis to downstream processing (Zhang et al 2012).…”

Section: Introductionmentioning

confidence: 99%

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Specialization of theDrosophilanuclear export family protein, Nxf3, for piRNA precursor export

Kneuss

Munafò

Eastwood

et al. 2019

Preprint

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The piRNA pathway is a conserved, small RNA-based immune system that protects animal germ cell genomes from the harmful effects of transposon mobilisation. In Drosophila ovaries, most piRNAs originate from dual-strand clusters, which generate piRNAs from both genomic strands. Dual-strand clusters use non-canonical transcription mechanisms. Although transcribed by RNA polymerase II, cluster transcripts lack splicing signatures and poly(A) tails. mRNA processing is important for general mRNA export mediated by Nuclear export factor 1. Although UAP56, a component of the transcription and export complex, has been implicated in piRNA precursor export, it remains unknown how dual-strand cluster transcripts are specifically targeted for piRNA biogenesis by export from the nucleus to cytoplasmic processing centers. Here we report that dual-strand cluster transcript export requires CG13741/Bootlegger and the Drosophila Nuclear export factor family protein, Nxf3. Bootlegger is specifically recruited to piRNA clusters and in turn brings Nxf3. We find that Nxf3 specifically binds to piRNA precursors and is essential for their export to piRNA biogenesis sites, a process that is critical for germline transposon silencing. Our data shed light on how dual-strand clusters compensate for a lack of canonical features of mature mRNAs to be specifically exported via Nxf3, ensuring proper piRNA production.Kneuss et al., page 3 We thank Martin H. Fabry for help with computational analyses. We thank the CRUK Cambridge Institute Bioinformatics, Genomics, Microscopy, Research Instrumentation and Cell Services, and Proteomics Core Facilities for technical support. We thank the University of Cambridge Department of Genetics Fly Facility for microinjection services and fly stock generation. We thank the Vienna Drosophila Resource Center and the Bloomington Stock Center for fly stocks. We thank Julius Brennecke for anti-Piwi, anti-Aub and anti-Ago3 antibodies, William Theurkauf for anti-Rhi antibody and fly strains, Elisa Izaurralde for anti-Nxt1 antibody, and Paul Lasko for anti-UAP56 antibody.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Specialization of theDrosophilanuclear export family protein, Nxf3, for piRNA precursor export

Kneuss

Munafò

Eastwood

et al. 2019

Preprint

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“…Export of mRNA from the nucleus requires the association of the heterodimeric nuclear RNA export receptor Nxf1-Nxt1 (Tap-p15) with mRNA cargo, which is enabled by the nuclear RNA helicase UAP56/Hel25E and the associated THO complex, present from yeast to human (Heath et al, 2016;Kohler and Hurt, 2007;Luo et al, 2001;Strasser and Hurt, 2001). In the Drosophila germline, both, UAP56 and the THO complex are enriched at Rhino-dependent piRNA clusters and are required for efficient piRNA production from these loci (Hur et al, 2016;Zhang et al, 2012;Zhang et al, 2018). To ask whether Nxf1-Nxt1 might also export germline piRNA precursors, we generated flies expressing Nxf1 or Nxt1 with a 'localization and affinitypurification' (LAP) tag consisting of GFP and a triple FLAG peptide.…”

Section: Nxf3 Localizes To Pirna Transcription and Processing Sitesmentioning

confidence: 99%

A heterochromatin-specific RNA export pathway facilitates piRNA production

ElMaghraby

Andersen

Pühringer

et al. 2019

Preprint

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Interactive data links: High-throughput sequencing (UCSC browser session); Mass spectrometry (interactive plots) PIWI-interacting RNAs (piRNAs) guide transposon silencing in animals. The 22-30nt piRNAs are processed in the cytoplasm from long non-coding RNAs. How piRNA precursors, which often lack RNA processing hallmarks of export-competent transcripts, achieve nuclear export is unknown. Here, we uncover the RNA export pathway specific for piRNA precursors in the Drosophila germline. This pathway requires Nxf3-Nxt1, a variant of the hetero-dimeric mRNA export receptor Nxf1-Nxt1. Nxf3 interacts with UAP56, a nuclear RNA helicase essential for mRNA export, and CG13741/Bootlegger, which recruits Nxf3-Nxt1 and UAP56 to heterochromatic piRNA source loci. Upon RNA cargo binding, Nxf3 achieves nuclear export via the exportin Crm1, and accumulates together with Bootlegger in perinuclear nuage, suggesting that after export, Nxf3-Bootlegger delivers precursor transcripts to the piRNA processing sites. Our findings indicate that the piRNA pathway bypasses nuclear RNA surveillance systems to achieve export of heterochromatic, unprocessed transcripts to the cytoplasm, a strategy also exploited by retroviruses.

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“…Formation of these "mini-clusters", through an uncharacterized epigenetic mechanism, provides an independent adaptation mechanism. In Drosophila, germline piRNA clusters and transposon mini-clusters are bound by the RDC complex, which is composed of the HP1 homolog Rhino (Rhi), which co-localizes with the linker protein Deadlock (Del) and the Rai1 homolog Cutoff (Cuff) (Chang et al, 2019;Chen et al, 2016; Le Thomas et al, 2014;Mohn et al, 2014;Pane et al, 2011;Parhad et al, 2017;Yu et al, 2015;Zhang et al, 2018;Zhang et al, 2014). The three components of the RDC are co-dependent for localization to clusters, and essential to germline piRNA production.…”

Section: Introductionmentioning

confidence: 99%

Adaptive evolution targets a piRNA precursor transcription network

Parhad

Zhang

et al. 2019

Preprint

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In Drosophila, transposon-silencing piRNAs are derived from heterochromatic clusters and a subset of euchromatic transposon insertions, which are transcribed from internal non-canonical initiation sites and flanking canonical promoters. Rhino binds to Deadlock, which recruits TRF2 to promote non-canonical transcription of these loci. Cuff co-localizes with Rhino and Del. The role of Cuff is less well understood, but the cuff gene shows hallmarks of adaptive evolution, which frequently targets functional interactions within host defense systems. We show that Drosophila simulans cuff is a dominant negative allele when expressed in Drosophila melanogaster, where it traps Deadlock, TRF2 and the transcriptional co-repressor CtBP in stable nuclear complexes. Cuff promotes Rhino and Deadlock localization, driving non-canonical transcription. CtBP, by contrast, suppresses canonical cluster and transposon transcription, which interferes with downstream non-canonical transcription and piRNA production. Cuff, TRF2 and CtBP thus form a network that balances canonical and non-canonical piRNA precursor transcription.

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Co-dependent Assembly of Drosophila piRNA Precursor Complexes and piRNA Cluster Heterochromatin

Cited by 33 publications

References 55 publications

Specialization of theDrosophilanuclear export family protein, Nxf3, for piRNA precursor export

Specialization of theDrosophilanuclear export family protein, Nxf3, for piRNA precursor export

A heterochromatin-specific RNA export pathway facilitates piRNA production

Adaptive evolution targets a piRNA precursor transcription network

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