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1.
Nature ; 622(7982): 402-409, 2023 Oct.
Article in English | MEDLINE | ID: mdl-37758951

ABSTRACT

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.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Endoribonucleases , Piwi-Interacting RNA , Animals , Argonaute Proteins/metabolism , Caenorhabditis elegans/enzymology , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/chemistry , Caenorhabditis elegans Proteins/metabolism , DNA Transposable Elements/genetics , Endoribonucleases/chemistry , Endoribonucleases/metabolism , Holoenzymes/chemistry , Holoenzymes/metabolism , Piwi-Interacting RNA/chemistry , Piwi-Interacting RNA/genetics , Piwi-Interacting RNA/metabolism , RNA Cap Analogs/chemistry , RNA Cap Analogs/metabolism
2.
Genes Dev ; 35(17-18): 1304-1323, 2021 09 01.
Article in English | MEDLINE | ID: mdl-34413138

ABSTRACT

Piwi-interacting RNAs (piRNAs) constitute a class of small RNAs that bind PIWI proteins and are essential to repress transposable elements in the animal germline, thereby promoting genome stability and maintaining fertility. C. elegans piRNAs (21U RNAs) are transcribed individually from minigenes as precursors that require 5' and 3' processing. This process depends on the PETISCO complex, consisting of four proteins: IFE-3, TOFU-6, PID-3, and ERH-2. We used biochemical and structural biology approaches to characterize the PETISCO architecture and its interaction with RNA, together with its effector proteins TOST-1 and PID-1. These two proteins define different PETISCO functions: PID-1 governs 21U processing, whereas TOST-1 links PETISCO to an unknown process essential for early embryogenesis. Here, we show that PETISCO forms an octameric assembly with each subunit present in two copies. Determination of structures of the TOFU-6/PID-3 and PID-3/ERH-2 subcomplexes, supported by in vivo studies of subunit interaction mutants, allows us to propose a model for the formation of the TOFU-6/PID-3/ERH-2 core complex and its functionality in germ cells and early embryos. Using NMR spectroscopy, we demonstrate that TOST-1 and PID-1 bind to a common surface on ERH-2, located opposite its PID-3 binding site, explaining how PETISCO can mediate different cellular roles.


Subject(s)
Caenorhabditis elegans Proteins , Caenorhabditis elegans , Animals , Argonaute Proteins/genetics , Argonaute Proteins/metabolism , Caenorhabditis elegans/genetics , Caenorhabditis elegans/metabolism , Caenorhabditis elegans Proteins/metabolism , DNA Transposable Elements , Germ Cells/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism
3.
Int J Mol Sci ; 21(14)2020 Jul 15.
Article in English | MEDLINE | ID: mdl-32679873

ABSTRACT

Poly-(ADP-ribosyl)-ation (PARylation) is a reversible post-translational modification of proteins and DNA that plays an important role in various cellular processes such as DNA damage response, replication, transcription, and cell death. Here we designed a fully genetically encoded fluorescent sensor for poly-(ADP-ribose) (PAR) based on Förster resonance energy transfer (FRET). The WWE domain, which recognizes iso-ADP-ribose internal PAR-specific structural unit, was used as a PAR-targeting module. The sensor consisted of cyan Turquoise2 and yellow Venus fluorescent proteins, each in fusion with the WWE domain of RNF146 E3 ubiquitin ligase protein. This bipartite sensor named sPARroW (sensor for PAR relying on WWE) enabled monitoring of PAR accumulation and depletion in live mammalian cells in response to different stimuli, namely hydrogen peroxide treatment, UV irradiation and hyperthermia.


Subject(s)
Bacterial Proteins/analysis , Fluorescence Resonance Energy Transfer/methods , Fluorescent Dyes/analysis , Luminescent Proteins/analysis , Poly Adenosine Diphosphate Ribose/analysis , Bacterial Proteins/genetics , Biosensing Techniques/methods , Cell Line , Fluorescent Dyes/metabolism , Humans , Luminescent Proteins/genetics , Open Reading Frames , Protein Domains , Recombinant Fusion Proteins/analysis , Recombinant Fusion Proteins/genetics , Ubiquitin-Protein Ligases/analysis , Ubiquitin-Protein Ligases/genetics
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