Drosophila PAF1 Modulates PIWI/piRNA Silencing Capacity

Clark, Josef P.; Rahman, Reazur; Yang, Nachen; Yang, Linda; Lau, Nelson C.

doi:10.1016/j.cub.2017.07.052

Cited by 21 publications

(22 citation statements)

References 48 publications

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“…In addition to Eggless‐KD, H1‐KD and HP1‐KD also had no impact on the silencing by Piwi–piRNA and λN‐Nxf2 in the reporter system based on plasmids (Figs EV3F and EV4G–J). While this result is consistent with a report of a study using a plasmid‐based reporter assay to detect Piwi‐directed silencing in Drosophila ovary somatic sheet cells (Clark et al , ), it is at odds with previous reports demonstrating that the methyltransferase Eggless is required for Panx‐mediated silencing using the tethering system in the Drosophila ovary (Sienski et al , ; Yu et al , ). These conflicting results are probably due to the different expression systems of λN fusion proteins.…”

Section: Resultssupporting

confidence: 91%

“…Recently, it was reported that Pol II‐associated proteins, PAF1 and RTF1, antagonize Piwi‐directed silencing (Clark et al , ), which are factors involved in the modulation of promoter release, elongation, and termination of Pol II (Chen et al , ; Van Oss et al , ; Vos et al , ). The Panx–Nxf2–P15 complex might interfere with Pol II via the PAF1 complex, considering that fission yeast PAF1 represses AGO1/siRNA‐directed silencing (Kowalik et al , ).…”

Section: Discussionmentioning

confidence: 99%

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Nuclear RNA export factor variant initiates piRNA‐guided co‐transcriptional silencing

et al. 2019

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The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade proteins in Drosophila, Piwi transcriptionally silences its targets through interactions with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by H3K9me3 and H1. Here, we identified Nxf2, a nuclear RNA export factor (NXF) variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-P15 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of the target reporter in a manner independent of H3K9me3 marks or H1. However, continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with target TE transcripts in a Piwidependent manner. These findings suggest a model in which the Panx-Nxf2-P15 complex enforces the association of Piwi with target transcripts to trigger co-transcriptional repression, prior to heterochromatin formation in the nuclear piRNA pathway. Our results provide an unexpected connection between an NXF variant and small RNA-mediated co-transcriptional silencing. The EMBO JournalKensaku Murano et al F Density plots for normalized H3K9me3 ChIP-seq signals over the consensus sequence from mdg1, gypsy (targeted by Piwi-piRNA, orange), and roo (not targeted by Piwi-piRNA, blue) TEs in EGFP-, Piwi-, Panx-, and Nxf2-KD OSCs. G Boxplots as in (D) showing fold changes in the H3K9me3 levels of Piwi-piRNA-targeted and un-targeted TEs upon the indicated KD. H Northern blotting for Idefix-piRNA, traffic jam (tj)-piRNA, and esiRNA sl-1 (control) on total RNA isolated from OSCs with the indicated KD.

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Section: Resultssupporting

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Nuclear RNA export factor variant initiates piRNA‐guided co‐transcriptional silencing

et al. 2019

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“…In addition to Eggless-KD, H1-KD and HP1-KD also had no impact on the silencing by Piwi–piRNA and λN-Nxf2 in the reporter system based on plasmids (Figures S3F and S4G-J). While this result is consistent with a report of a study using a plasmid-based reporter assay to detect Piwi-directed silencing in Drosophila ovary somatic sheet cells (Clark et al, 2017), it is at odds with previous reports demonstrating that the methyltransferase Eggless is required for Panx-mediated silencing using the tethering system in the Drosophila ovary (Sienski et al, 2015; Yu et al, 2015). These conflicting results are probably due to the different expression systems of λN fusion proteins.…”

Section: Resultssupporting

confidence: 91%

Nuclear RNA export factor variant initiates piRNA-guided co-transcriptional silencing

Murano

Iwasaki

Ishizu

et al. 2019

Preprint

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1The PIWI-interacting RNA (piRNA) pathway preserves genomic integrity by 2 repressing transposable elements (TEs) in animal germ cells. Among PIWI-clade 3 proteins in Drosophila, Piwi transcriptionally silences its targets through interactions 4 with cofactors, including Panoramix (Panx) and forms heterochromatin characterized by 5H3K9me3 and H1. Here, we identified Nxf2, a nuclear RNA export factor (NXF) 6 variant, as a protein that forms complexes with Piwi, Panx, and p15. Panx-Nxf2-p15 7 complex formation is necessary in the silencing by stabilizing protein levels of Nxf2 8 and Panx. Notably, ectopic targeting of Nxf2 initiates co-transcriptional repression of 9 the target reporter in a manner independent of H3K9me3 marks or H1. However, 10 continuous silencing requires HP1a and H1. In addition, Nxf2 directly interacts with 11 target TE transcripts in a Piwi-dependent manner. These findings suggest a model in 12 which the Nxf2-Panx-p15 complex enforces the association of Piwi with target 13

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“…Small RNA libraries were sequenced as 75bp single end reads on the NextSeq550. Adapters for the small RNA libraries were removed with CutAdapt and then mapped to the Drosophila transposon consensus sequences from RepBase and Flybase using Bowtie v1 with 1 mismatch and R plotting scripts as applied in our previous published studies on Drosophila piRNAs (Clark et al, 2017; Sytnikova et al, 2014).…”

Section: Methodsmentioning

confidence: 99%

Har-P, ashortP-element variant, weaponizesP-transposase to severely impairDrosophiladevelopment

Srivastav

Rahman

et al. 2019

Preprint

Self Cite

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Har-P, a short P-element variant, weaponizes P-transposase to1 severely impair Drosophila development.Running title: Har-P and P-transposase drive gonadal dysgenesis. 15 16 ABSTRACT 1 Without transposon-silencing Piwi-interacting RNAs (piRNAs), transposition 2 causes an ovarian atrophy syndrome in Drosophila called gonadal dysgenesis (GD).3Harwich (Har) strains with P-elements cause severe GD in F1 daughters when Har 4 fathers mate with mothers lacking P-element-piRNAs (i.e. ISO1 strain). To address the 5 mystery of why Har induces severe GD, we bred hybrid Drosophila with Har genomic 6 fragments into the ISO1 background to create HISR-D or HISR-N lines that still cause 7 Dysgenesis or are Non-dysgenic, respectively. In these lines, we discovered a highly 8 truncated P-element variant we named "Har-P" as the most frequent de novo insertion. 9Although HISR-D lines still contain full-length P-elements, HISR-N lines lost functional 10 P-transposase but retained Har-P's that when crossed back to P-transposase restores 11 GD induction. Finally, we uncovered P-element-piRNA-directed repression on Har-P's 12 transmitted paternally to suppress somatic transposition. The Drosophila short Har-P's 13 and full-length P-elements relationship parallels the MITEs/DNA-transposase in plants 14 and SINEs/LINEs in mammals. 15 16 17 GD retention and loss in hybrid Drosophila lines with reduced P-element copy numbers.1 To genetically isolate the causative transposon strongly inducing GD and 2 facilitate discovery by whole genome sequencing (WGS), we generated hybrid lines 3 where only a minor fraction of the Har genome is within the background of the ISO1 4 reference genome sequence. We first conducted several fertility-permissive 5 backcrosses between female Har and male ISO1, selecting hybrid progeny that 6 propagated a red-eye phenotype which we attributed to the "red" eyes due to Har alleles 7 replacing the cn, bw, sp, alleles on Chromosome 2R (Chr2R) of ISO1 ( Fig. 2A-abridged 8 scheme, Fig. 2-S1-detailed scheme). We then performed an initial GD validation screen 9 with many vials of individual hybrid males crossed to ISO1 females and selecting for 10 lines that caused 100% GD from this cross. Lines were propagated with additional self-11 crosses and further in-bred with single-sibling pairs. We then subjected multiple 12 independent Har-ISO1-Selfed-Red (HISR) lines to a second GD assay. Finally, we 13 conducted qPCR to identify the lines with the greatest reduction of P-element copy 14 numbers ( Fig. 2B) and settled on 4 lines each either retaining severe paternally-induced 15 GD (HISR-D) or had lost this capacity (HISR-N) ( Fig. 2C). 16Genomic PCR genotyping of deletion loci of Har compared to ISO1 in HISR lines 17 indicated that these lines carried mostly ISO1 genomes ( Fig. 2-S2). Therefore, we 18 performed WGS of the parental Har and ISO1 strains, the 8 HISR lines, and the pi[2] 19 and Birmingham (Birm) strains, two classic strains with similar numbers of P-elements 20 but diametric capacity to induce GD (Engels et al., ...

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Drosophila PAF1 Modulates PIWI/piRNA Silencing Capacity

Cited by 21 publications

References 48 publications

Nuclear RNA export factor variant initiates piRNA‐guided co‐transcriptional silencing

Nuclear RNA export factor variant initiates piRNA‐guided co‐transcriptional silencing

Nuclear RNA export factor variant initiates piRNA-guided co-transcriptional silencing

Har-P, ashortP-element variant, weaponizesP-transposase to severely impairDrosophiladevelopment

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