piRNA processing by a trimeric Schlafen-domain nuclease

Transposable elements are genomic parasites that expand within and spread between genomes1. PIWI proteins control transposon activity, notably in the germline2,3. These proteins recognize their targets through small RNA co-factors named PIWI-interacting RNAs (piRNAs), making piRNA biogenesis a key specificity-determining step in this crucial genome immunity system. Although the processing of piRNA precursors is an essential step in this process, many of the molecular details remain unclear. Here, we identify an endoribonuclease, precursor of 21U RNA 5′-end cleavage holoenzyme (PUCH), that initiates piRNA processing in the nematode Caenorhabditis elegans. Genetic and biochemical studies show that PUCH, a trimer of Schlafen-like-domain proteins (SLFL proteins), executes 5′-end piRNA precursor cleavage. PUCH-mediated processing strictly requires a 7-methyl-G cap (m7G-cap) and a uracil at position three. We also demonstrate how PUCH interacts with PETISCO, a complex that binds to piRNA precursors4, and that this interaction enhances piRNA production in vivo. The identification of PUCH concludes the search for the 5′-end piRNA biogenesis factor in C. elegans and uncovers a type of RNA endonuclease formed by three SLFL proteins. Mammalian Schlafen (SLFN) genes have been associated with immunity5, exposing a molecular link between immune responses in mammals and deeply conserved RNA-based mechanisms that control transposable elements.

Sequencing data is available at NCBI's Sequence Read Archive under accession number PRJNA925182 (https://dataview.ncbi.nlm.nih.gov/object/PRJNA925182? reviewer=951lavsn8a0umpj5j17m8bk738).The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE72 partner repository with the dataset identifier PXD039502.Username: reviewer_pxd039502@ebi.ac.ukPassword: T2C6anBX Coordinates and structure factors of the TOFU-6eTUDOR TOFU-1pep complex structure have been deposited in the Protein Data Bank with accession codes PDB ID 8BY5.Wormbase WS289 was used in this study.Uniprot was regularly used.Because of constant updates specific versions cannot be given.Always the most recent version was used.Alphafold database (https://alphafold.ebi.ac.uk/) was used in this work.

Field-specific reporting
Please select the one below that is the best fit for your research.If you are not sure, read the appropriate sections before making your selection.

Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative.

Replication
All gels and blots were done in duplicate.
All protein analyses were done in duplicate.
Sequencing data were obtained from one experiment based on biological triplicates.In our experience this yields very robust results.Many papers build on duplicates only.The tofu-2(e216a) mutant was analyzed twice in this manner.Mass spectrometry is based on one experiment that uses biological quadruplicates.This is widely accepted practice in the field of quantitative proteomics.
Imaging results were based on observations on different individuals animals.At least ten animals were imaged and a representative image was used in the manuscript.Images of cells were obtained with two different transfection experiments.Representative images of cells are shown in the manuscript.Seven cells were imaged in both experiments.
All PUCH cleavage reactions were done in duplicate.The ligation experiment was done in triplicate.Cleavage of the 10 nucleotide substrate and the gel-shift with the 5'P end were the only experiments performed once.In all cases replication yielded equivalent results.
Randomization Randomization is not relevant to this study, as distinct genotypes and protein preparations had to be generated before each experiment.

Blinding
Blinding was used in the initial analysis of the sRNA data.The bio-informatician received only strain names without genotype information.
After initial analysis, results were interpreted in light of the genotypes, so at this stage no blinding was applied.This is also not possible if questions are derived from the initial results of analysis.
Blinding was applied in the in vitro PUCH activity assays from lysates of transfected BmN4 cells.Lysate sources were unknown to the N. Podvalnaya when running the cleavage assays.In all other experiments blinding was not used in experiments for the following reasons: The primary results of our work were rather objective, making blinding not needed.
The explorative nature of the study makes blinding impossible and rather unlikely to affect the results.Genotypes of animals needed to be established before analysis.
Reporting for specific materials, systems and methods Anti-Actin (Sigma, #A5060).Quality control by Sigma-Aldrich: working dilutions for western blot of at least 1:250 were determined using rat brain or chicken muscle extracts IRDye® 800CW Goat anti-Mouse IgG Secondary Antibody (LI-COR, #926-32210): Isolation of specific antibodies was accomplished by affinity chromatography using pooled mouse IgG covalently linked to agarose.Based on ELISA and flow cytometry, this antibody reacts with the heavy and light chains of mouse IgG1, IgG2a, IgG2b, and IgG3, and with the light chains of mouse IgM and IgA.This antibody was tested by dot blot and and/or solid-phase adsorbed for minimal cross-reactivity with human, rabbit, goat, rat, and horse serum proteins, but may cross-react with immunoglobulins from other species.The conjugate has been specifically tested and qualified for Western blot applications.

nature portfolio | reporting summary
March 2021 IRDye® 680LT Donkey anti-Rabbit IgG (LI-COR, #926-68023).The antibody was isolated from antisera by immunoaffinity chromatography using antigens coupled to agarose beads.Based on immune electrophoresis, this antibody reacts with the heavy chains of rabbit IgG, and with the light chains common to most rabbit immunoglobulins.No reactivity was detected against nonimmunoglobulin serum proteins.This antibody was tested by ELISA and/or solid-phase adsorbed to ensure minimal cross-reactivity with bovine, chicken, goat, guinea pig, Syrian hamster, horse, human, mouse, rat, and sheep serum proteins, but may cross-react with immunoglobulins from other species.The conjugate has been specifically tested and qualified for Western blot applications.
Worm work: Specificity tested by absence of signal in lysates from untagged strains.Monoclonal anti-HA (clone 12CA5) antibody: Soluble lysate from exponentially-growing yeast cultures expressing HA-tagged proteins were separated on 4-15% gradient gel (BioRad).Proteins were blotted an a nitrocellulose membrane using semi-dry transfer (10 min High MW program on TransBlot Turbo blotter) and the membrane was blocked 1h in 5% skim milk/PBS/0.1% Tween-20.Primary antibodies: mouse anti-HA (in-house or Covance, 1:1000 o/N in blocking solution) at 4°C Secondary antibody: goat anti-mouse HRPcoupled (BIORAD 170-5047) 1:3000 1h at RT in blocking solution.The western blot was developed using ECL substrate Dura (Thermo, #34076).Exposure times as indicated in the figure.The experiment was performed by Katharina Bender from Brian Luke's group, IMB, Mainz, Germany.anti-Histone H3 antibody: By immunoblotting, a working antibody dilution of 1:5,000-1:10,000 is recommended using a whole cell extract of the A431 human epidermoid carcinoma cell line, and a whole cell extract of the mouse fibroblast NIH3T3 cell line.By immunoblotting, a working antibody dilution of 1:2,500-1:5,000 is recommended using a whole cell extract of the rat pheochromocytoma PC12 cell line.

Mycoplasma contamination
Not tested.
Policy information about studies involving human research participants and Sex and Gender in Research.information on the approval of the study protocol must also be provided in the manuscript.
anti-mouse IgG, HRP-linked antibody: Application Key: WB-Western Blot IP-Immunoprecipitation IHC-Immunohistochemistry ChIP-Chromatin Immunoprecipitation IF-Immunofluorescence F-Flow Cytometry E-P-ELISA-Peptide.Species Cross-Reactivity Key: H-Human M-Mouse R-Rat Hm-Hamster Mk-Monkey Vir-Virus Mi-Mink C-Chicken Dm-D. melanogaster X-Xenopus Z-Zebrafish B-Bovine Dg-Dog Pg-Pig Sc-S.cerevisiae Ce-C.elegans Hr-Horse All-All Species Expected anti-rabbit IgG, HRP-linked antibody: Application Key: WB-Western Blot IP-Immunoprecipitation IHC-Immunohistochemistry ChIP-Chromatin Immunoprecipitation IF-Immunofluorescence F-Flow Cytometry E-P-ELISA-Peptide.Species Cross-Reactivity Key: H-Human M-Mouse R-Rat Hm-Hamster Mk-Monkey Vir-Virus Mi-Mink C-Chicken Dm-D. melanogaster X-Xenopus Z-Zebrafish B-Bovine Dg-Dog Pg-Pig Sc-S.cerevisiae Ce-C.elegans Hr-Horse All-All Species Expected Monoclonal anti-MYC: no signal in non-tagged C. elegans strains; signal at expected MW in tagged strains only.Eukaryotic cell lines Policy information about cell lines and Sex and Gender in Research Cell line source(s) BmN4 cells used in this study were provided to us by Dr. Ramesh Pillai, University of Geneva, in 2015.BmN4 cells originate from Kyushu University, Dr. Kusakabe.Also see: https://www.cellosaurus.org/CVCL_Z634Authentication BmN4 cells have not been authenticated.
We require information from authors about some types of materials, experimental systems and methods used in many studies.Here, indicate whether each material, system or method listed is relevant to your study.If you are not sure if a list item applies to your research, read the appropriate section before selecting a response.