A nuclear RNA export factor variant licenses piRNA-guided heterochromatin formation
Small RNA silencing pathways protect genome integrity in part through establishing heterochromatin at transposon loci. In animals, this process requires piRNA-guided targeting of nuclear PIWI proteins to nascent transcripts. The molecular events contributing to heterochromatin formation upon PIWI binding to nascent RNA, a transient molecule at chromatin, are unknown. Here, we identify SFINX, a protein complex that is required for Piwi-mediated co-transcriptional silencing in Drosophila. It consists of Nxf2—a variant of the nuclear RNA export factor Nxf1/Tap, the mRNA export co-factor Nxt1/p15, and the Piwi-associated protein Panoramix. In the absence of Nxf2, Panoramix is targeted for degradation and piRNA-loaded Piwi is unable to establish heterochromatin. Consequently, nxf2 mutants exhibit severe transposon de-repression and are sterile. We show that within SFINX, Panoramix connects to the heterochromatin machinery while Nxf2 enables target silencing via nascent RNA. Thus, the Nxf2-Nxt1 heterodimer—despite having originated from core mRNA export machinery—has been repurposed for heterochromatin formation. Our data establish an unexpected link between nuclear small RNA biology and NXF-variants, which are widespread in animal lineages, but mostly lack ascribed functions.
Sample Processing Protocol
OSCs were collected after trypsinization by centrifugation, washed with PBS and centrifuged again. The cell pellet was resuspended in LB1 (10 mM Tris-HCl pH=7.5, 2 mM MgCl2, 3 mM CaCl2, freshly supplemented with Complete Protease Inhibitor Cocktail (Roche)), incubated at 4 °C for 10 min followed by a centrifugation step. The pellet was resuspended in LB2 (10 mM Tris-HCl pH=7.5, 2 mM MgCl2, 3 mM CaCl2, 0,5 % IGEPAL CA-630, 10 % glycerol, freshly supplemented with Complete Protease Inhibitor Cocktail (Roche)), incubated at 4 °C for 10 min followed by a centrifugation step. The isolated nuclei were lysed in LB3 (50 mM Tris-HCl pH=8, 150 mM NaCl, 2 mM MgCl2, 0,5 % Triton X-100, 0,25 % IGEPAL CA-630, 10 % glycerol, freshly supplemented with Complete Protease Inhibitor Cocktail (Roche)), incubated at 4 °C for 20 min followed by a centrifugation step. Nuclear lysate was used for immunoprecipitation with Flag M2 Magnetic Beads (Sigma) for 2 h at 4 °C. The beads were washed three times for 10 min with LB3 and six times for 1 min in MS Wash Buffer (20 mM Tris-HCl pH=8, 150 mM NaCl). Samples were processed by on-bead digest with LysC on the beads, eluted with glycine followed by trypsin digestion. This was analyzed using an UltiMate 3000 RSLCnano System (Thermo Fisher Scientific) coupled to a Q Exactive HF mass spectrometer (Thermo Fisher Scientific), equipped with a Proxeon nanospray source (Thermo Fisher Scientific). Peptides were loaded onto a trap column (Thermo Fisher Scientific, PepMap C18, 5 mm × 300 μm ID, 5 μm particles, 100 Å pore size) at a flow rate of 25 μL/min using 0.1% TFA as mobile phase. After 10 min, the trap column was switched in line with the analytical column (Thermo Fisher Scientific, PepMap C18, 500 mm × 75 μm ID, 2 μm, 100 Å). Peptides were eluted using a flow rate of 230 nl/min and a binary 3h gradient. The gradient starts with the mobile phases: 98% A (water/formic acid, 99.9/0.1, v/v) and 2% B (water/acetonitrile/formic acid, 19.92/80/0.08, v/v/v), increases to 35%B over the next 180 min, followed by a gradient in 5 min to 90%B, stays there for 5 min and decreases in 2 min back to the gradient 98%A and 2%B for equilibration at 30°C. The Q Exactive HF mass spectrometer was operated in data-dependent mode, using a full scan (m/z range 380-1500, nominal resolution of 60,000, target value 1E6) followed by MS/MS scans of the 10 most abundant ions. MS/MS spectra were acquired using normalized collision energy of 27, isolation width of 1.4 m/z, resolution of 30.000 and the target value was set to 1E5. Precursor ions selected for fragmentation (exclude charge state 1, 7, 8, >8) were put on a dynamic exclusion list for 60 s. Additionally, the minimum AGC target was set to 5E3 and intensity threshold was calculated to be 4.8E4. The peptide match feature was set to preferred and the exclude isotopes feature was enabled. for XLMS: Panx (263-446) with an N-terminal Twin-Strep-tag, Nxf2 with an N-terminal His6-tag and Nxt1 with an N-terminal FLAG-tag were expressed in Trichoplusia ni High5 cells. After cell-lysis in lysis buffer (LB) (50 mM Tris-HCl pH=8, 150 mM NaCl, 0,05 % TX100, 1mM DDT) freshly supplemented with Complete Protease Inhibitor Cocktail (Roche) and with Benzonase (~10U/ml) for 30 min at 4 °C and the lysate was cleared by centrifugation. For purification, a StrepTactin Superflow HC resin (IBA GmbH) was used with the AKTA Purifier FPLC system. The bound protein complex was eluted with LB supplemented with 5 mM desthiobiotin. After affinity purification, the complex containing fractions were pooled and further purified by SEC using a Superdex 200 10/300 (GE Healthcare) in SEC buffer (SB) (50 mM Tris-HCl pH=8, 150 mM NaCl, 1mM DDT). The purified complex was cross-linked with 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride (DMTMM), with a final concertation of 4 mM. Complex and cross-linker were reacted for 30 minutes at room temperature. To stop the reaction, Tris-HCl pH=8 was added to a final concentration of 100 mM. Samples were processed by in-solution digest with trypsin. The digested samples were enriched for cross-links (XLs) prior to LC-MS/MS analysis using SEC. Therefore, approx. 15 µg of the digest was separated on a TSKgel SuperSW2000 column (300 mm × 4.5 mm × 4 μm, Tosoh Bioscience). The three high mass fractions were collected and measured on the mass spectrometer. Full scans were recorded at a resolution of 120000 ranging from 350-1600 m/z (AGC 1e6, 60 ms max. injection time). The top 10 most intense ions with charge state >3+ from the full scan were selected for fragmentation. MS/MS spectra were recorded at 30,000 resolution (AGC 5e4, max. injection time 150 ms, isolation width 1.0 m/z). DMTMM cross-links were fragmented with higher energy C-trap dissociation (HCD) using a stepped collision energy of 30-33-35%. Once a precursor was selected for an MS/MS scan, it was excluded from fragmentation for 30 s.
Data Processing Protocol
For peptide identification, the RAW-files were loaded into Proteome Discoverer (version 184.108.40.206, Thermo Scientific). All hereby created MS/MS spectra were searched using MSAmanda v220.127.116.1149, Engine version v18.104.22.16849 (Dorfer V. et al., J. Proteome Res. 2014 Aug 1;13(8):3679-84). For the first step search the RAW-files were searched against Drosophila melanogaster reference translations retrieved from Flybase (dmel_all-translation-r6.13; 21,983 sequences; 20,112,742 residues), using the following search parameters: The peptide mass tolerance was set to ±5 ppm and the fragment mass tolerance to 15ppm. The maximal number of missed cleavages was set to 2. The result was filtered to 1 % FDR on protein level using Percolator algorithm integrated in Thermo Proteome Discoverer. A sub-database was generated for further processing. For the second step the RAW-files were searched against the created sub-database, using the following search parameters: Beta-methylthiolation on cysteine was set as a fixed modification, oxidation on methionine, deamidation on asparagine and glutamine, acetylation on lysine, phosphorylation on serine, threonine and tyrosine, methylation and di-methylation on lysine and arginine, tri-methylation on lysine, ubiquitination on lysine were set as variable modifications. Monoisotopic masses were searched within unrestricted protein masses for tryptic enzymatic specificity. The peptide mass tolerance was set to ±5 ppm and the fragment mass tolerance to ±15 ppm. The maximal number of missed cleavages was set to 2. The result was filtered to 1% FDR on peptide level using Percolator algorithm integrated in Thermo Proteome Discoverer. The localization of the post-translational modification sites within the peptides was performed with the tool ptmRS, based on the tool phosphoRS (Taus T. et al., J. Proteome Res. 2011, 10, 5354-62). Peptide areas have been quantified using in-house-developed tool APQuant: http://ms.imp.ac.at/index.php?action=peakjuggler (publication under review). Data Processing Protocol for XLMS: Raw files were analyzed with pLink (Version 2.3.3, S.-B. Fan et al., Curr. Protoc. Bioinforma (2015) p. 8.21.1-8.21.19) using the settings as described above. Used cross-linker: DMTMM (-18.0116 Da, reactivity towards lysine, protein N-terminus, serine, threonine and tyrosine or aspartate, glutamate and the protein C-terminus, respectively); MS1 accuracy: 10 ppm; MS2 accuracy: 20 ppm; used enzyme: trypsin; max. missed cleavages: 4; minimum peptide length: 5; max. modifications: 4; static modifications: carbamidomethylation (cysteine, +57.021 Da); dynamic modifications: oxidation (methionine, +15.995 Da). For the database search a database containing the three cross-linked proteins was used and the false discovery rate (FDR) was set to 1%. To reduce the number of false positives, XLs were manually validated. For XL visualization, xiNET was used (C. W. Combe et al., Mol. Cell. Proteomics 2015, 14, 1137–47).