Germ granule compartments coordinate specialized small RNA production

Abstract

Germ granules are biomolecular condensates present in most animal germ cells. One function of germ granules is to help maintain germ cell totipotency by organizing mRNA regulatory machinery, including small RNA-based gene regulatory pathways. TheC. elegansgerm granule is compartmentalized into multiple subcompartments whose biological functions are largely unknown. Here, we identify a new subcompartment of theC. elegansgerm granule, which we term the E compartment. The E compartment is nonrandomly positioned within the germ granule. We identified five proteins that localize to the E compartment, including the RNA-dependent RNA polymerase (RdRP) EGO-1, the Dicer-related helicase DRH-3, the Tudor domain-containing protein EKL-1, and two intrinsically disordered proteins, EGC-1 and ELLI-1. Localization of EGO-1 to the E granule enables synthesis of a specialized class of 22G RNAs, which derive exclusively from 5’regions of a subset of germline-expressed mRNAs. Defects in E compartment assembly elicit disordered production of endogenous siRNAs, which disturbs fertility and the RNAi response. Our results define a new subcompartment of theC. elegansgerm granule and suggest that one function of germ granule compartmentalization is to facilitate the localized production of specialized classes of small regulatory RNAs.

Compartmentalized localization of perinuclear proteins within germ granules inC. elegans

“…In C. elegans , many of the factors involved in the biogenesis of small regulatory RNAs, including siRNAs and piRNAs, as well as the Argonaute (AGO) proteins that bind these small RNAs, are largely enriched in germ granules 21 . We and other groups previously showed that the factors localized in E granules and Mutator foci contribute to the production of distinct cohorts of siRNAs using non-overlapping RNA templates 27,33,34 . piRNAs, which are 21 nt in length and start with 5′-monophosphorylated uracil, are produced from Pol II-transcribed piRNA precursors, exported to the cytoplasm, and subjected to a series of processing steps in the germline (Fig.…”

“…S1B), suggesting that the fluorescence-tagged proteins largely recapitulated their endogenous functions. We further collected published nematode strains expressing fluorescence-tagged perinuclear proteins, which were constructed previously in our laboratory 27,48 . In total, we collected a nematode strain library that consisted of more than 70 strains covering approximately 60 genes (Table S1).…”

“…The Z granule contains ZNFX-1 and WAGO-4, which promote the transgenerational inheritance of RNAi 14,31,32 . The E granule and Mutator foci are two independent subcompartments required for 22G RNA generation using a largely nonoverlapping set of RNA transcripts as templates 27,33,34 . The SIMR-1 foci, which are marked by SIMR-1, promotes siRNA amplification from piRNA targets and drive small RNA specificity for the nuclear Argonaute protein HRDE-1 35,36 .…”

“…ZNFX-1, WAGO-4, PID-2/ZSP-1 and LOTR-1 accumulate in the Z granule, promoting RNAi inheritance and piRNA-induced gene silencing 14,15,43,44 . Approximately 90 C. elegans proteins have been found to be enriched in germ granules through a variety of methods, including proteomic approaches, such as TurboID/MS and IP/MS technologies 21,27,36,45,46 . Although high-resolution imaging and genetic analysis help us determine the sub-organelle localization of some perinuclear proteins, the precise localization of a considerable portion of these proteins within germ granules is still unclear.…”

“…Germ granules are widely present in the germ cells of a variety of animals including worms, flies, zebrafish, Xenopus and mice, and are spatially organized into multilayered structures [21][22][23][24][25] . In C. elegans, recent studies have suggested that the germ granule is subcompartmentalized into several distinct regions that enclose particular sets of proteins, including P granule, Z granule, E granule, Mutator foci, SIMR foci and P-bodies 21,26,27 . The P granule was the first identified membraneless organelle to form via LLPS, which exhibits multiple liquid-like behaviors and has emerged as a leading model for the study of biomolecular condensates 28,29 .…”