Channel nuclear pore complex subunits are required for transposon silencing in Drosophila
- Cancer Research UK Cambridge Institute, University of Cambridge, Li Ka Shing Centre, United Kingdom
- Department of Life Science, Faculty of Health, Education and Life Sciences, Birmingham City University, United Kingdom
- School of Biosciences, College of Life and Environmental Sciences, University of Birmingham, United Kingdom
- Birmingham Center for Genome Biology, University of Birmingham, United Kingdom
Figures
Figure 1 with 3 supplements
Requirement of Nuclear Pore Complex (NPC) subunits for gene regulation and transposon control.
(A) Heatmap summarising literature data for selected Nups and export factors and (B) effects of each knockdown (kd) on cell viability, transposon expression levels with respect to siGFP and Yb-body …
Figure 1—figure supplement 1
Yb-body formation is dependent on flam.
(A) Schematic of a Drosophila ovariole, the functional unit of the fly ovary. The follicle cells enclosing egg chambers are highlighted in magenta and cells of the germline are shown in grey. (B) …
Figure 1—figure supplement 2
Effects of Nuclear Pore Complex subunits on transposable element (TE) silencing and Yb-body assembly in ovarian somatic cells (OSCs).
(A) Fold-changes in steady-state RNA levels of the indicated transposons or protein-coding mRNAs in OSCs upon the indicated knockdowns (kd) for 96 or 48 hrs. Values are relative to siGFP control kd …
Figure 1—figure supplement 3
Effects of Nuclear Pore Complex subunits on Yb-body assembly in ovarian somatic cells (OSCs).
Confocal images of Yb and Piwi proteins and polyA-tail containing transcripts (oligo-dT) in OSCs upon the indicated knockdown. All knockdowns are 96 hr unless indicated otherwise.
Figure 2 with 3 supplements
Phenylalanine-glycine (FG) nucleoporins Nup54 and Nup58 function specifically in transposable element (TE) silencing.
(A) Cartoon showing the Nuclear Pore Complex (NPC) structure with nuclear and cytosolic factors involved in flam export; inset shows the domain structure of the Nup62 complex subunits and of …
Figure 2—figure supplement 1
Effects of Nup54 and Nup58 knockdown (kd) on flam expression.
(A) Volcano plots showing fold-changes and corresponding significance of genes and transposable elements (TEs) in RNA-seq from the indicated kd. Red: germline TEs; yellow: TEs active in somatic and …
Figure 2—figure supplement 2
Nup54 and Nup58 are required for PIWI-interacting RNA (piRNA) production from flam.
(A) Bar plots showing small RNA size profiles of transposable element (TE)-mapped reads upon the indicated knockdowns (kd). Counts are normalised to 1 million mapped miRNAs. Red: antisense; blue: sen…
Figure 2—figure supplement 3
Nup54 and Nup58 are required for flam export.
(A) Confocal images of flam RNA and polyadenylated mRNAs (oligo-dT) upon the indicated knockdown (kd). Arrowheads indicate cytosolic flam foci. (B) Schematic of the probes used for flam DNA-FISH. (C,…
Figure 3 with 2 supplements
Nup54 and Nup58 are specifically required for flam export and processing into PIWI-interacting RNAs (piRNAs).
(A) Schematic of the Nup54 and Nup58 domain structure and the deletion constructs used in rescue experiments and co-immunoprecipitation assays. Green box: phenylalanine-glycine (FG)-repeats; purple …
Figure 3—figure supplement 1
Effect of nup54MB mutants on gene expression.
(A) Fold-changes in steady-state RNA levels of the gypsy transposon or act5c gene in ovarian somatic cells (OSCs) nucleofected with the indicated siRNAs and rescue constructs. Values are relative to …
Figure 3—figure supplement 2
Effect of nup54MB mutants on Yb-body assembly.
Confocal images of Piwi and Yb proteins in ovaries of the indicated genotypes. Quantification of the size of Yb-bodies in the indicated genotypes is shown to the right. ndots = number of Yb-bodies …
Figure 4 with 1 supplement
Nup54 and Nup58 coordinate export and licensing of flam via Nxf1 and Yb.
(A, B) Volcano plots showing enrichment and corresponding significance of biotinylated proteins identified via proximity labelling, followed by mass spectrometry (PL-MS) from ovarian somatic cells …
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Figure 4—source data 1
Source data for volcano plot shown in Figure 4A.
- https://cdn.elifesciences.org/articles/66321/elife-66321-fig4-data1-v2.txt
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Figure 4—source data 2
Source data for volcano plot shown in Figure 4B.
- https://cdn.elifesciences.org/articles/66321/elife-66321-fig4-data2-v2.txt
Figure 4—figure supplement 1
Nup54/Nup58 coordinate flam export and licensing by bridging Nxf1 and Yb.
(A) Coverage plot of the indicated CLIP-seq reads over the first 50 kb of the flam locus (n = 3). The mappability for 50 bp reads is shown at the bottom. (B) Volcano plot showing enrichment and …
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Figure 4—figure supplement 1—source data 1
Source data for volcano plot shown in Figure 4—figure supplement 1B.
- https://cdn.elifesciences.org/articles/66321/elife-66321-fig4-figsupp1-data1-v2.txt
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Drosophila melanogaster) | fs(1)Yb | FlyBase | FBgn0000928 | |
| Gene (Drosophila melanogaster) | flamenco | FlyBase | FBgn0267704 | |
| Gene (Drosophila melanogaster) | Nup54 | FlyBase | FBgn0033737 | |
| Gene (Drosophila melanogaster) | Nup58 | FlyBase | FBgn0038722 | |
| Gene (Drosophila melanogaster) | Elys | FlyBase | FBgn0031052 | |
| Gene (Drosophila melanogaster) | Nup43 | FlyBase | FBgn0038609 | |
| Gene (Drosophila melanogaster) | Nup214 | FlyBase | FBgn0010660 | |
| Gene (Drosophila melanogaster) | Nup62 | FlyBase | FBgn0034118 | |
| Gene (Drosophila melanogaster) | Nup93-1 | FlyBase | FBgn0027537 | |
| Gene (Drosophila melanogaster) | Nup98-96 | FlyBase | FBgn0039120 | |
| Gene (Drosophila melanogaster) | Nxf1/Sbr | FlyBase | FBgn0003321 | |
| Gene (Drosophila melanogaster) | Nxt1 | FlyBase | FBgn0028411 | |
| Gene (Drosophila melanogaster) | lamin | FlyBase | FBgn0002525 | |
| Gene (Drosophila melanogaster) | Nup107 | FlyBase | FBgn0027868 | |
| Gene (Drosophila melanogaster) | Nup154 | FlyBase | FBgn0021761 | |
| Gene (Drosophila melanogaster) | Zuc | FlyBase | FBgn0261266 | |
| Gene (Drosophila melanogaster) | tj | FlyBase | FBgn0000964 | |
| Antibody | Anti-Piwi (Rabbit polyclonal) | DOI:10.1016/j.cell.2007.01.043 | IF(1:500) WB(1:5000) | |
| Antibody | Anti-Yb (Mouse monoclonal) | DOI:10.1101/gad.1989510 | IF(1:500) WB(1:1000) | |
| Antibody | Anti-Yb (Rabbit polyclonal) | DOI:10.1038/emboj.2011.308 | WB(1:1000) | |
| Antibody | Anti-tubulin (Rabbit polyclonal) | Abcam | Cat# ab18251, RRID:AB_2210057 | WB(1:5000) |
| Antibody | Anti-HA (Rabbit monoclonal) | Cell Signaling Technology | Cat# 3724, RRID:AB_1549585 | WB(1:1000) |
| Antibody | Anti-HA (Rabbit polyclonal) | Abcam | Cat# ab9110, RRID:AB_307019 | WB(1:1000) |
| Antibody | Anti-FLAG (Mouse monoclonal) | Sigma | Cat# F1804, RRID:AB_262044 | WB(1:1000) |
| Antibody | Anti-lamin (Mouse monoclonal) | Developmental Studies Hybridoma Bank | Cat# adl67.10, RRID:AB_528336 | IF(1:200) |
| Antibody | Anti-Nuclear Pore Complex Proteins (Mouse monoclonal) | Biolegend | Cat# 902907, RRID:AB_2565026 | IF(1:500) |
| Antibody | Anti-Mouse IgG Alexa Fluor-488 (Goat polyclonal) | Thermo Fisher Scientific | Cat# A-11029, RRID:AB_2534088 | IF(1:500) |
| Antibody | Anti-Rabbit IgG Alexa Fluor-647 (Goat polyclonal) | Thermo Fisher Scientific | Cat# A-21245, RRID:AB_2535813 | IF(1:500) |
| Antibody | GFP-Booster Atto-488 (Alpaca monoclonal) | Chromotek | Cat# gba488-100, RRID:AB_2631386 | IF(1:500) |
| Commercial assay or kit | IRDye 800CW Streptavidin | LI-COR | Cat# 926-32230 | WB(1:4000) |
| Commercial assay or kit | Streptavidin, Alexa Fluor 555 Conjugate | Thermo Fisher Scientific | Cat# S-21381 | IF(1:500) |
| Commercial assay or kit | Mouse monoclonal anti-FLAG M2 magnetic beads | Sigma-Aldrich | Cat# CatM8823, RRID:AB_2637089 | |
| Commercial assay or kit | Dynabeads MyOne Streptavidin C1 | Thermo Fisher Scientific | Cat# 65001 | |
| Commercial assay or kit | Pierce IP Lysis Buffer-100 ml | Thermo Fisher Scientific | Cat# 87787 | |
| Commercial assay or kit | RIPA Lysis and Extraction Buffer | Thermo Fisher Scientific | Cat# 89901 | |
| Commercial assay or kit | Thermo Scientific Pierce anti-HA magnetic beads | Thermo Fisher Scientific | Cat# 88836 | |
| Commercial assay or kit | cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail | Sigma-Aldrich | Cat# 11836170001 | |
| Commercial assay or kit | RNasin Plus RNase Inhibitor | Promega | Cat# N2615 | |
| Commercial assay or kit | Effectene Transfection Reagent | Qiagen | Cat# 301427 | |
| Commercial assay or kit | Nucleofector Kit V | Lonza | Cat# VVCA-1003 | |
| Commercial assay or kit | DNase I, Amplification Grade | Thermo Fisher Scientific | Cat# 18068015 | |
| Commercial assay or kit | RNaseOUT Recombinant Ribonuclease Inhibitor | Thermo Fisher Scientific | Cat# 10777019 | |
| Commercial assay or kit | Magne HaloTag Beads,20% Slurry | Promega | Cat# G7282 | |
| Commercial assay or kit | Deoxynucleotide Solution Set (100 mM; 25 μmol each) | New England Biolabs | Cat# N0446S | |
| Commercial assay or kit | SuperScript III Reverse Transcriptase | Thermo Fisher Scientific | Cat# 18080085 | |
| Commercial assay or kit | Chloroform anhydrous 99+% | Sigma-Aldrich | Cat# 288306 | |
| Commercial assay or kit | TRIzol Reagent | Thermo Fisher Scientific | Cat# 15596026 | |
| Commercial assay or kit | Insulin solution human | Sigma-Aldrich | Cat# I9278 | |
| Commercial assay or kit | Fibronectin from human plasma 0.1% solution | Sigma-Aldrich | Cat# F0895 | |
| Commercial assay or kit | ProLong Diamond Antifade Mountant | Thermo Fisher Scientific | Cat# P36961 | |
| Commercial assay or kit | Fly Extract | Drosophila Genomics Resource Center | Cat# 1645670 | |
| Commercial assay or kit | SMARTer RNA Unique Dual Index Kit – 24U | Clontech | Cat# 634451 | |
| Commercial assay or kit | RiboPOOL 10 nM for Drosophila | Cambridge Bioscience | ||
| Commercial assay or kit | D-Biotin solution | Thermo Fisher Scientific | Cat# B20656 | |
| Commercial assay or kit | Shields and Sang M3 Insect Medium | Sigma | Cat# S3652 | |
| Commercial assay or kit | Library Quantification Kit | Kapa Biosystems | Cat# KK4873 | |
| Commercial assay or kit | ProTEV Plus | Promega | Cat# V6101 | |
| Commercial assay or kit | Paraformaldehyde, 16% w/v aq. soln., methanol free | Alfa Aesar | Cat# 043368.9M | |
| Commercial assay or kit | RNase A (affinity purified)1 mg/ml | Thermo Fisher Scientific | Cat# AM2271 | |
| Commercial assay or kit | NuPAGE 4–12%, Bis-Tris,1.5 mm, Mini Protein Gel, 10-well | Thermo Fisher Scientific | Cat# NP0335BOX | |
| Commercial assay or kit | Xfect Transfection Reagent | Takara Bio | Cat# 631318 | |
| Commercial assay or kit | Pierce Protein A/G Magnetic Beads | Thermo Fisher Scientific | Cat# 88802 | |
| Commercial assay or kit | Agencourt RNAClean XP beads | Beckman Coulter | Cat# A63987 | |
| Commercial assay or kit | NEBNext Ultra Directional RNA Library Prep Kit for Illumina | New England Biolabs | Cat# E7420L | |
| Commercial assay or kit | SMARTer Stranded RNA-Seq Kit | Takara Bio | Cat# 634839 | |
| Commercial assay or kit | RNeasy Mini Kit | Qiagen | Cat# 74104 | |
| Commercial assay or kit | Cell Line Nucleofector kit V | Lonza | Cat# VVCA-1003 | |
| Commercial assay or kit | Nucleofector II device | Lonza | Cat# AAB-1001 | |
| Cell line (Drosophila melanogaster) | S2 cells | Thermo Fisher Scientific | Cat# R69007, RRID:CVCL_Z232 | |
| Cell line (Drosophila melanogaster) | Ovarian somatic cells (OSCs) | DOI:10.1038/nature08501 | RRID:CVCL_IY73 | |
| Software, algorithm | Fiji | ImageJ | RRID:SCR_002285 | |
| Software, algorithm | Proteome Discoverer 2.1 | Thermo Fisher Scientific | RRID:SCR_014477 | |
| Software, algorithm | STAR | DOI:10.1093/bioinformatics/bts635 | RRID:SCR_015899 | |
| Software, algorithm | DEseq2 | DOI:10.1186/s13059-014-0550-8 | RRID:SCR_015687 | |
| Software, algorithm | Image Studio Lite | LI-COR | RRID:SCR_013715 |
Additional files
-
Source code 1
Custom script for Yb-bodies analysis in Fiji.
- https://cdn.elifesciences.org/articles/66321/elife-66321-code1-v2.zip
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Source code 2
Custom script for flam RNA-FISH analysis in Fiji.
- https://cdn.elifesciences.org/articles/66321/elife-66321-code2-v2.zip
-
Supplementary file 1
List of siRNA sequences, qPCR primers, and fly stocks used in this study.
- https://cdn.elifesciences.org/articles/66321/elife-66321-supp1-v2.xlsx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/66321/elife-66321-transrepform-v2.pdf
