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      Caenorhabditis elegans germ granules are present in distinct configurations that differentially associate with RNAi-targeted RNAs

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      1 , 2 , 1 , 1
      bioRxiv
      Cold Spring Harbor Laboratory

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          Abstract

          RNA silencing pathways are complex, highly conserved, and perform widespread, critical regulatory roles. In C. elegans germlines, RNA surveillance occurs through a series of perinuclear germ granule compartments—P granules, Z granules, SIMR foci, and Mutator foci—multiple of which form via phase separation and exhibit liquid-like properties. The functions of individual proteins within germ granules are well-studied, but the spatial organization, physical interaction, and coordination of biomolecule exchange between compartments within germ granule “nuage” is less understood. Here we find that key proteins are sufficient for compartment separation, and that the boundary between compartments can be reestablished after perturbation. Using super-resolution microscopy, we discover a toroidal P granule morphology which encircles the other germ granule compartments in a consistent exterior-to-interior spatial organization. Combined with findings that nuclear pores primarily interact with P granules, this nuage compartment organization has broad implications for the trajectory of an RNA as it exits the nucleus and enters small RNA pathway compartments. Furthermore, we quantify the stoichiometric relationships between germ granule compartments and RNA to reveal discrete populations of nuage that differentially associate with RNAi-targeted transcripts, possibly suggesting functional differences between nuage configurations. Together, our work creates a more spatially and compositionally accurate model of C. elegans nuage which informs the conceptualization of RNA silencing through different germ granule compartments.

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          Most cited references56

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          Enzymatic assembly of DNA molecules up to several hundred kilobases.

          We describe an isothermal, single-reaction method for assembling multiple overlapping DNA molecules by the concerted action of a 5' exonuclease, a DNA polymerase and a DNA ligase. First we recessed DNA fragments, yielding single-stranded DNA overhangs that specifically annealed, and then covalently joined them. This assembly method can be used to seamlessly construct synthetic and natural genes, genetic pathways and entire genomes, and could be a useful molecular engineering tool.
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            Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans.

            Experimental introduction of RNA into cells can be used in certain biological systems to interfere with the function of an endogenous gene. Such effects have been proposed to result from a simple antisense mechanism that depends on hybridization between the injected RNA and endogenous messenger RNA transcripts. RNA interference has been used in the nematode Caenorhabditis elegans to manipulate gene expression. Here we investigate the requirements for structure and delivery of the interfering RNA. To our surprise, we found that double-stranded RNA was substantially more effective at producing interference than was either strand individually. After injection into adult animals, purified single strands had at most a modest effect, whereas double-stranded mixtures caused potent and specific interference. The effects of this interference were evident in both the injected animals and their progeny. Only a few molecules of injected double-stranded RNA were required per affected cell, arguing against stochiometric interference with endogenous mRNA and suggesting that there could be a catalytic or amplification component in the interference process.
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              Germline P granules are liquid droplets that localize by controlled dissolution/condensation.

              In sexually reproducing organisms, embryos specify germ cells, which ultimately generate sperm and eggs. In Caenorhabditis elegans, the first germ cell is established when RNA and protein-rich P granules localize to the posterior of the one-cell embryo. Localization of P granules and their physical nature remain poorly understood. Here we show that P granules exhibit liquid-like behaviors, including fusion, dripping, and wetting, which we used to estimate their viscosity and surface tension. As with other liquids, P granules rapidly dissolved and condensed. Localization occurred by a biased increase in P granule condensation at the posterior. This process reflects a classic phase transition, in which polarity proteins vary the condensation point across the cell. Such phase transitions may represent a fundamental physicochemical mechanism for structuring the cytoplasm.
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                Author and article information

                Journal
                bioRxiv
                BIORXIV
                bioRxiv
                Cold Spring Harbor Laboratory
                25 May 2023
                : 2023.05.25.542330
                Affiliations
                [1 ]Department of Biological Sciences, University of Southern California, Los Angeles, CA 90089
                [2 ]Present address: Departments of Developmental Biology and Genetics, Stanford University School of Medicine, Stanford CA, 94305
                Author notes
                [*]

                These authors contributed equally

                Corresponding author: Carolyn M. Phillips, Department of Biological Sciences, University of Southern California, 1050 Childs Way RRI 201, Los Angeles, CA 90089-2910. cphil@ 123456usc.edu
                Article
                10.1101/2023.05.25.542330
                10246010
                37292702
                877a3f1a-378c-4240-a864-73c2efc43007

                This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.

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