Abstract
Membraneless organelles are sites for RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. How small ncRNAs utilise phase separated environments for their function is unclear. We investigated how the PIWI-interacting RNA (piRNA) pathway engages with the membraneless organelle P granule in Caenorhabditis elegans. Proteomic analysis of the PIWI protein PRG-1 reveals an interaction with the constitutive P granule protein DEPS-1. DEPS-1 is not required for piRNA biogenesis but piRNA-dependent silencing: deps-1 mutants fail to produce the secondary endo-siRNAs required for the silencing of piRNA targets. We identify a motif on DEPS-1 which mediates a direct interaction with PRG-1. DEPS-1 and PRG-1 form intertwining clusters to build elongated condensates in vivo which are dependent on the Piwi-interacting motif of DEPS-1. Additionally, we identify EDG-1 as an interactor of DEPS-1 and PRG-1. Our study reveals how specific protein-protein interactions drive the spatial organisation and piRNA-dependent silencing within membraneless organelles.
Highlights
Membraneless organelles are sites for RNA biology including small non-coding RNA mediated gene silencing
The C. elegans PIWI-interacting RNA (piRNA) pathway offers a unique model for understanding how membraneless organelles engage with small RNA pathways as it requires proteins that can localise to two distinct and juxtaposed biomolecular condensates to achieve gene repression: the perinuclear P granules where PRG-1 resides[24] and the secondary endo-siRNAs are entirely dependent on the mutator foci[29,30,31]
To identify proteins that intersect biomolecular condensate functions and small RNA pathways we performed immunoprecipitation (IP) of PRG-1 followed by mass spectrometry (Supplementary Fig. S1a)
Summary
Membraneless organelles are sites for RNA biology including small non-coding RNA (ncRNA) mediated gene silencing. Recent studies reveal that membraneless organelles can be formed by proteins and nucleic acids condensing out of the bulk intracellular milieu, giving rise to liquid- or gel-like environments[1,2,3] These phase separated organelles, formed by proteins and nucleic acids, are sites for different aspects of eukaryotic RNA biology: the nucleolus is required for the assembly of ribosomes[4], stress granules allow for translational stalling of mRNAs during stressresponse[5] and processing bodies (P-bodies) organise small RNAmediated regulation of mRNA6. The C. elegans piRNA pathway offers a unique model for understanding how membraneless organelles engage with small RNA pathways as it requires proteins that can localise to two distinct and juxtaposed biomolecular condensates to achieve gene repression: the perinuclear P granules where PRG-1 resides[24] and the secondary endo-siRNAs are entirely dependent on the mutator foci[29,30,31]
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