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The nascent RNA binding complex SFiNX licenses piRNA-guided heterochromatin formation

Abstract

The PIWI-interacting RNA (piRNA) pathway protects genome integrity in part through establishing repressive heterochromatin at transposon loci. Silencing requires piRNA-guided targeting of nuclear PIWI proteins to nascent transposon transcripts, yet the subsequent molecular events are not understood. Here, we identify SFiNX (silencing factor interacting nuclear export variant), an interdependent protein complex required for Piwi-mediated cotranscriptional silencing in Drosophila. SFiNX consists of Nxf2–Nxt1, a gonad-specific variant of the heterodimeric messenger RNA export receptor Nxf1–Nxt1 and the Piwi-associated protein Panoramix. SFiNX mutant flies are sterile and exhibit transposon derepression because piRNA-loaded Piwi is unable to establish heterochromatin. Within SFiNX, Panoramix recruits heterochromatin effectors, while the RNA binding protein Nxf2 licenses cotranscriptional silencing. Our data reveal how Nxf2 might have evolved from an RNA transport receptor into a cotranscriptional silencing factor. Thus, NXF variants, which are abundant in metazoans, can have diverse molecular functions and might have been coopted for host genome defense more broadly.

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Fig. 1: The Nxf2–Nxt1 heterodimer interacts with Panoramix.
Fig. 2: Nxf2 is required for Piwi-mediated heterochromatin formation.
Fig. 3: Targeting Nxf2 to nascent RNA induces cotranscriptional silencing.
Fig. 4: Panoramix and Nxf2–Nxt1 form the interdependent SFiNX complex.
Fig. 5: Panoramix-mediated silencing via nascent RNA requires Nxf2.
Fig. 6: Nxf2 lost nucleoporin binding.
Fig. 7: A key role for Nxf2’s RNA binding unit within SFiNX.

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Data availability

All sequencing data used for this study (Supplementary Table 12) have been deposited at NCBI GEO (GSE120617). The mass spectrometry data have been deposited to the ProteomeXchange Consortium via PRIDE (PXD011201)84. Coordinate and structure factors of the UBA-linker-helix and the dmNxf2–Nxt1 complex are available from the Protein Data Bank (PDB 6OPF and 6MRK). Source data for Fig. 1a are available in Supplementary Table 1, Fig. 1f,g and Supplementary Fig. 1d,e in Supplementary Table 2, Fig. 2c and Supplementary Fig. 2d in Supplementary Table 3, Supplementary Fig. 2i,k in Supplementary Table 4 and Fig. 7d in Supplementary Table 6. Source data for Figs. 3d,e, 5h and 7f are available online.

Code availability

All custom code is based on the publicly available code used in ref. 64 with modifications indicated in the Methods section.

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Acknowledgements

We thank K. Meixner for experimental support, P. Duchek and J. Gokcezade for generating CRISPR edited and transgenic flies, the VBCF NGS unit for deep sequencing, VBCF Protein Technologies Facility for protein expression, VBCF VDRC unit for fly stocks, the MFPL monoclonal facility for antibodies and the TRiP and Bloomington stock centers for flies. We thank A. Koehler and G. Riddihough (both at www.lifescienceeditors.com) for comments on the manuscript. We thank the Brennecke laboratory, particularly P. Andersen, for support and feedback. The Brennecke laboratory is supported by the Austrian Academy of Sciences, the European Community (grant no. ERC-2015-CoG—682181) and the Austrian Science Fund (grant nos. F 4303 and W1207). J. Batki was supported by the Boehringer Ingelheim Fonds. X-ray diffraction studies were conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team beamlines, which are supported by NIGMS grant no. P30 GM124165 and US Department of Energy grant no. DE-AC02-06CH11357. The Pilatus 6 M detector on 24-ID-C beam line is funded by a NIH-ORIP HEI grant (no. S10 RR029205). MSKCC core facilities are supported by grant no. P30 CA008748. This work was supported by funds from NIH U19-CA179564 and the Maloris Foundation (to D.J.P.) and MSKCC core grant P30 CA008748.

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Authors and Affiliations

Authors

Contributions

J. Batki, J.S., V.I.A., L.L. and K.K. performed all molecular biology and fly experiments. D.H., J.S. and J. Batki performed the computational analyses, C.E.S. and K.M. performed the X-link mass spectrometry analysis. M.N. generated the phylogenetic comparisons of NXF proteins. J.W. generated, purified and grew crystals of the UBA-linker-helix and the Nxf2 NTF2l–Nxt1 complex, performed the X-ray crystallographic analyses and performed the SEC–MALS assay with W.X., under the supervision of D.J.P. The paper was written by J. Batki, J.S. and J. Brennecke with input from J.W. and D.J.P.

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Correspondence to Julius Brennecke.

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Integrated supplementary information

Supplementary Figure 1 Related to Fig. 1.

a, Average peptide peak intensities for indicated proteins in Panoramix or Nxf2 immunoprecipitates (data from experiment shown in Fig. 1a; respective peptide intensities measured in control samples were subtracted). b, Cartoon depicting frameshift positions caused by the guide RNA-induced insertions/deletions in the Nxf2 protein. c, Morphology of representative ovaries from flies of indicated genotypes (scale bar: 1mm). d, e, Scatter plots showing steady state ovarian RNA levels (transcripts per million) of genes (d) and transposons (e) in panx or piwi mutants compared to control. f, Confocal images of egg chambers (scale bar: 20 μm) from panoramix (left panel) or nxf2 (right panel) mutant flies co-stained for Panoramix or Nxf2, respectively, with Piwi. The remaining staining in the panoramix mutant egg chambers corresponds to a background staining in the surrounding muscle sheet.

Supplementary Figure 2 Related to Fig. 2.

a, Confocal images of ovarioles (scale bar: 20μm) from w[1118] control and nxf2 mutant flies stained for Piwi (inverted grayscale). b, Confocal images showing egg chambers (scale bar: 20μm) from flies expressing GFP-tagged Nxf2 in addition to indicated germline-specific gene knockdowns (inverted grayscale). c, Box plot showing fold change (compared to control knockdown) of piRNA levels in ovaries depleted of indicated genes specifically in the germline (box plot definition as in Fig. 2b). d, Volcano plot showing changes (as effect size) in steady state transposon levels from RNA-seq experiments (n=3 biological replicates). The plot compares piwi knockdown versus control knockdown. e-g, Density profiles showing normalized reads from H3K9me3 ChIP-seq (top), or RNA Pol II ChIP-seq (bottom) experiments mapping to the mdg1 (e), burdock (f) or F-element (g) transposon consensus sequences (knockdowns indicated). h, Box plots showing fold changes (compared to control knockdown) of H3K9me3 ChIP-seq signal (top) or input DNA (bottom) in 1kb tiles in OSCs depleted of indicated genes. Piwi dependent and independent regions are shown separately (box plot definition as in Fig. 2b). i, Volcano plots showing differential gene expression analysis from OSC RNA-seq experiments (gene knockdowns indicated; n=3 biological replicates). j, Western blots showing knockdown efficiencies of two different nxf2 siRNAs in OSCs. ATP5A served as loading control. k, Volcano plots showing differential expression analysis of genes (left) and transposons (right) from OSC RNA-seq experiments (gene knockdowns indicated; n=3 biological replicates). The plots compare the two tested independent siRNAs targeting nxf2. Source data: panel d, i, k: Supplementary Table 3, 4; uncropped blot images: Supplementary Data Set 1.

Supplementary Figure 3 Related to Fig. 3.

a, Bar graph showing genomic copy number of indicated genes based on digital droplet PCR. Two clonal reporter OSC lines were analyzed and clone 1, which harbors a single insertion, was used throughout the study. b, Box plots showing the λN-control normalized GFP intensity in OSCs at indicated days after transfection of λN-tagged Panoramix protein; n=2500 cells; box plot definition as in Fig. 3b). c-f, Western blots showing levels of indicated FLAG-tagged fusion proteins (λN or Gal4) in OSC lysates (related to Fig. 3c, f, g, h; Actin served as loading control). Source data: uncropped blot images: Supplementary Data Set 1.

Supplementary Figure 4 Related to Fig. 4.

a, Bar graph showing fold change of RNA-seq levels of indicated genes in mutant ovaries compared to control ovaries (n=1). b, Confocal images showing OSCs (scale bar: 10μm) with indicated, transiently transfected FLAG-tagged Panoramix constructs. c, Left: Shown is the entire size exclusion chromatogram (Fig. 4f) of the affinity-purified Strep-Panoramix eluate (mAU = milli-absorbance unit). To the right, an SDS-PAGE of the 7 peak fractions from the second detected peak is shown (Coomassie blue staining), indicating that this peak does not contain protein. d, Calibration curve of the HiLoad 16/60 Superdex 200 size exclusion chromatography column. Grey dots indicate protein standards based on which the calibration curve was calculated (black dashed line). The recombinant SFiNX complex is indicated based on the elution volume in Fig. 4f. (V0: void volume of the column; Ve: measured elution volume; Mw: molecular weight.) Source data: uncropped gel images: Supplementary Data Set 1.

Supplementary Figure 5 Related to Fig. 5.

a-c, Western blot analysis of GFP or GFP-Nxf2 immunoprecipitation experiments using lysate from S2 cells transiently co-transfected with indicated FLAG-Panoramix expressing plasmids (relative amount loaded in immunoprecipitation lanes: 3x). d, Protein sequence alignment of Panoramix (308-446) where the experimentally tested residues are marked in blue. Shown below are predicted secondary structural elements and the conservation score for each position. e, Helical wheel representation of the predicted amphipathic α-helix (322-339) within Panoramix. The point mutations introduced to abolish the Nxf2 interaction are indicated in purple color. f, Western blot analysis of lysate from S2 cells transiently transfected with indicated FLAG-Panoramix expressing plasmids. Actin served as loading control. g, Box plots showing GFP intensity in S2 cells 2 days after transfection with plasmids expressing indicated fusion proteins or empty control (numbers indicate fold-change in median GFP intensity normalized to GFP-only control; box plot definition as in Fig. 3b). h, Western blot analysis of immunoprecipitation experiments (bait: stabilized degron mutant GFP-Panoramix) using lysate from S2 cells transiently co-transfected with indicated FLAG-Nxf2 expressing plasmids (relative amount loaded in immunoprecipitation lanes: 3x). i, Co-purification of His-SUMO-Panoramix helix with untagged Nxf2 UBA domain without linker by two rounds of Ni-NTA affinity purification. Source data: uncropped blot and gel images: Supplementary Data Set 1.

Supplementary Figure 6 Related to Fig. 5.

a, Cartoon view of four UBA-linker-helix monomers in the crystal asymmetric unit. The linkers between the UBA domain and the Panoramix helix are largely invisible and are shown as dashed lines. b, Molecular weight of UBA-linker-Panoramix helix measured by SEC-MALS assay. The red line represents the SEC-MALS calculated molecular weight, which is 12.01±0.41 kDa. The theoretical molecular weight is 11.06 kDa, indicating that UBA-linker-Panoramix helix is a monomer in solution. c, Protein sequence alignment of the Nxf2 and Nxf1 UBA domains from indicated insect species. The relevant mutated residues are marked in red. Shown below are predicted secondary structural elements and the conservation score for each position. d, Western blot analysis of GFP-Nxf2 or GFP-Nxf1 immunoprecipitation experiments using lysate from S2 cells transiently co-transfected with indicated FLAG-Panoramix expressing plasmids (relative amount loaded in immunoprecipitation lanes: 3x). e, Left: Confocal images showing OSCs (scale bar: 10 μm) with indicated, transiently transfected FLAG-tagged, siRNA-resistant Panoramix constructs. Right: Western blots showing levels of indicated proteins in OSC lysates with indicated knockdowns and transiently transfected FLAG-tagged, siRNA-resistant Panoramix constructs (ATP5A served as loading control). f, Left: Confocal images showing OSCs (scale bar: 10 μm) with indicated, transiently transfected FLAG-tagged, siRNA-resistant Nxf2 constructs. Right: Western blots showing levels of indicated proteins in OSC lysates with indicated knockdowns and transiently transfected FLAG-tagged, siRNA-resistant Nxf2 constructs (ATP5A served as loading control). g, h, Western blots showing levels of indicated fusion proteins (left: λN, right: Gal4) in lysates of transiently transfected OSCs (related to Fig. 5i, j; Actin served as loading control). Source data: uncropped blot images: Supplementary Data Set 1.

Supplementary Figure 7 Related to Fig. 6.

Front and back views of the crystal structure of dmNxf2’s NTF2-like domain (purple and green) in complex with dmNxt1 (yellow and orange). Two NTF2-like domain–Nxt1 heterodimers were observed in the crystal asymmetric unit due to crystal packing. Invisible loops in the structure are shown as dashed curves.

Supplementary Figure 8 Related to Fig. 7.

a, Binding curve for the EMSA calculated from the phosphorimage shown in Fig. 7b; n=3; error bars: s.d.). b, Protein sequence alignment of the RRM/LRR domain of hsNXF1 and the first RRM/LRR domain of dmNxf2 (1st unit). Identical residues are highlighted by red squares. The key hsNXF1 residues involved in CTE RNA binding are indicated by blue triangles; red triangles mark residues which were mutated in dmNxf2 for the EMSA assay in Supplementary Fig. 8d. c, Coomassie stained SDS-PAGE showing the recombinant 1st unit of Nxf2, its point mutant variant, and the GB1 control peptide. d, Phosphorimage showing an Electrophoretic Mobility Shift Assay (EMSA) with labeled single stranded RNA (ssRNA) and increasing amount of the purified Nxf2 protein, its point mutant variant, and the GB1 control peptide. e, Confocal images depicting somatic cells of egg chambers (scale bar: 20 μm) from flies with indicated genotypes. Expression of the rescue transgenes was tested by GFP fluorescence, transposon derepression was assessed by mdg1 FISH. f, Western blots showing levels of indicated proteins in OSC lysates with indicated knockdowns and transiently transfected, siRNA-resistant rescue constructs (relates to experiment shown in Fig. 7e; ATP5A served as loading control). g, Western blots showing levels of indicated λN-fusion proteins in OSC lysates (related to Fig. 7g; Actin served as loading control). h, Maximum likelihood phylogenetic tree of NXF sequences from indicated species inferred with iqtree. Numbers represent ultrafast bootstrap branch support values. Scale bar indicates expected number of substitutions per codon site. Source data: uncropped blot and gel images: Supplementary Data Set 1.

Supplementary information

Supplementary Information

Supplementary Figs. 1–8, Supplementary Tables 6–13

Reporting Summary

Supplementary Table 1

co-IP-MS-volcano plot

Supplementary Table 2

RNA-seq-CRISPR_mutants

Supplementary Table 3

DGE-OSC-TE-table

Supplementary Table 4

DGE-OSC-gene-table

Supplementary Table 5

XL–MS

Supplementary Dataset 1

Uncropped blot and gel images

Supplementary Dataset 2

Flow cytometry gating strategy

Source data

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Batki, J., Schnabl, J., Wang, J. et al. The nascent RNA binding complex SFiNX licenses piRNA-guided heterochromatin formation. Nat Struct Mol Biol 26, 720–731 (2019). https://doi.org/10.1038/s41594-019-0270-6

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