mTOR Regulates Phase Separation of PGL Granules to Modulate Their Autophagic Degradation

Zhang, Gangming; Wang, Zheng; Du, Zhuo; Zhao, Hong

doi:10.1016/j.cell.2018.08.006

Cited by 181 publications

(165 citation statements)

References 32 publications

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“…We suggest that germ granules are not crucial for RNAi per se, but rather are important for small RNA homeostasis, with consequences that can persist for multiple generations. Previous studies have shown that the stability of the germ granules is affected by environmental changes (for instance temperature 55,56 ), and also that temperature shifts trigger changes in heritable small RNAs 3,57 . Because germ granules impact small RNA biogenesis, the granules could be important mediators in the translation of environmental changes to small RNA-mediated transgenerational memory.…”

Section: Discussionmentioning

confidence: 99%

Germ Granules Functions are Memorized by Transgenerationally Inherited Small RNAs

Lev

Toker

et al. 2019

Preprint

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In C. elegans nematodes, components of liquid-like germ granules were shown to be required for transgenerational small RNA inheritance. Surprisingly, we show here that mutants with defective germ granules (pptr-1, meg-3/4, pgl-1) can nevertheless inherit potent small RNA-based silencing responses, but some of the mutants lose this ability after many generations of homozygosity. Animals mutated in pptr-1, which is required for stabilization of P granules in the early embryo, display extremely strong heritable RNAi responses, which last for tens of generations, long after the responses in wild type animals peter out. The phenotype of mutants defective in the core germ granules proteins MEG-3 and MEG-4, depends on the genotype of the ancestors: Mutants that derive from maternal lineages that had functional MEG-3 and MEG-4 proteins exhibit enhanced RNAi inheritance for multiple generations. While functional ancestral meg-3/4 alleles correct, and even potentiates the ability of mutant descendants to inherit RNAi, defects in germ granules functions can be memorized as well; Wild type descendants that derive from lineages of mutants show impaired RNAi inheritance for many (>16) generations, although their germ granules are intact. Importantly, while P granules are maternally deposited, wild type progeny derived from meg-3/4 male mutants also show reduced RNAi inheritance. Unlike germ granules, small RNAs are inherited also from the sperm.Moreover, we find that the transgenerational effects that depend on the ancestral germ granules require the argonaute protein HRDE-1, which carries heritable small RNAs in the germline. Indeed, small RNA sequencing reveals imbalanced levels of many endogenous small RNAs in germ granules mutants. Strikingly, we find that hrde-1;meg-3/4 triple mutants inherit RNAi, although hrde-1 was previously thought to be essential for heritable silencing. We propose that germ granules sort and shape the RNA pool, and that small RNA inheritance memorizes this activity for multiple generations.

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Section: Discussionmentioning

confidence: 99%

Germ Granules Functions are Memorized by Transgenerationally Inherited Small RNAs

Lev

Toker

et al. 2019

Preprint

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“…protein with at least two multimerization domains) 1, 33 . Because PGLs are key assembly proteins for P-granules and can form granules on their own 6, 11, 24, 25 , we reasoned that PGLs use multiple self-interactions to drive granule assembly. We previously identified one dimerization domain (DD) centrally in PGL 27 , but DD missense mutations grossly affected protein stability.…”

Section: Resultsmentioning

confidence: 99%

“…Our work highlights the idea that interactions between structured regions are a driving force in vivo for assembly of liquid droplet granules as well as their biological function (see below). Recombinant PGL proteins make liquid droplets on their own in vitro 6, 11, 24 , suggesting that PGLs possess regions responsible for low affinity contacts in the full-length protein. We suggest that PGL uses both dimerization domains as well as additional low affinity contacts to facilitate liquid droplet formation.…”

Section: Discussionmentioning

confidence: 99%

“…P-granules are paradigmatic RNP granules 6, 7 with striking similarities to germ granules in Drosophila and vertebrates, both in subcellular location and composition 8 . They are crucial for fertility and totipotency 9 , and some of their components can display liquid droplet behavior 6, 10, 11 . In adult germ cells, P-granules localize to the cytoplasmic face of nuclear pores ( Figure 1A ) and contain both untranslated mRNAs 12 and numerous RNA regulatory proteins 13 .…”

Section: Introductionmentioning

confidence: 99%

“…PGL proteins self-assemble into granules, both in vitro using purified recombinant protein 24 and in intestinal nematode cells or mammalian cells in culture when expressed on their own 25, 26 . Artificial PGL granules display liquid droplet behavior 6, 11, 24 , indicating that PGL protein alone is sufficient to recapitulate the biophysical properties of P-granules in nematode cells 10 .…”

Section: Introductionmentioning

confidence: 99%

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Liquid droplet germ granules require assembly and localized regulators for mRNA repression

Aoki

Lynch

Crittenden

et al. 2018

Preprint

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20 21 Abbreviations: RNA, ribonucleic acid; UTR, untranslated region; GFP, green fluorescent 22 protein; CRISPR, Clustered Regularly Interspaced Short Palindromic Repeats; RNAi, 23 ribonucleic acid interference 24 25 Running title: P-granule assembly not sufficient for mRNA repression 128 cells expressing PGL-1::SNAP without λN22 possessed gfp RNAs in both nuclear and 129 cytoplasmic puncta (Figure S2B-E). We interpret nuclear puncta as nascent transcripts at 130 6 active transcription sites and cytoplasmic puncta as mRNAs, based on a previous study 31 . GFP 131 fluorescence was robust (Figure S2C,K), and PGL-1::SNAP localized to perinuclear granules 132 (Figure S2D), as in Figure 1. Germ cells expressing PGL-1::SNAP::λN22 lacked robust GFP 133 fluorescence (Figure S2G,K) and PGL-1::SNAP::λN22 localized to perinuclear granules (Figure 134 S2H), also as in Figure 1. The cytoplasmic RNA puncta were less diffuse with tethered PGL-1 135 than in the control and frequently colocalized with PGL-1::SNAP::λN22 in perinuclear granules 136 (Figure S2F-I,O; see Figure S3 for additional images). Imaging of the rachis, the shared 137 cytoplasmic space in the germline, did not reveal substantial aggregates of reporter RNA as 138 observed with mRNA in other studies 32 . The presence of gfp cytoplasmic transcripts (Figure 139 S2F) demonstrates that the reporter was not transcriptionally silenced, despite the lack of GFP 140 fluorescence. In summary, reporter RNA localized with PGL-1 when tethered and PGL-1 141 tethering repressed reporter protein expression. These results support the idea that P-granules 142 are sites of post-transcriptional repression. 143 144 Identification of the PGL N-terminal Dimerization Domain 145To test the significance of P-granule assembly to mRNA repression, we sought to perturb PGL-146 1 assembly into granules. An emerging principle is that multivalent-multivalent interactions drive 147 granule formation (e.g. protein with at least two multimerization domains) 1, 33 . Because PGLs 148are key assembly proteins for P-granules and can form granules on their own 6, 11, 24, 25 , we 149 reasoned that PGLs use multiple self-interactions to drive granule assembly. We previously 150 identified one dimerization domain (DD) centrally in PGL 27 , but DD missense mutations grossly 151 affected protein stability. We postulated the existence of another PGL multimerization domain 152 that might be more amenable to manipulation and again turned to structural analyses. 154The region N-terminal to DD has high sequence conservation (Figure S1B), which implies a 155 critical role in PGL function. Our initial efforts to express trypsin-mapped recombinant protein 157 disordered in the DD crystal structures 27 permitted robust expression sufficient for biochemical 158 and structural characterization (Figure S4A,B, see Methods for more details). Henceforth, we 159 refer to this stable protein fragment as the N-terminal dimerization domain (NtDD) (Figure 1B). 160We determined the C. japonica PGL-1 NtDD crystal structure to 1....

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Phase Separation: “The Master Key” to Deciphering the Physiological and Pathological Functions of Cells

Wei

et al. 2022

Advanced Biology

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Phase separation is a hot research field at present. It involves almost all aspects of cells and plays a significant role in cells, promising to be “a master key” in unlocking the mysteries of nature. In this review, the factors that affect phase separation are introduced, such as own component, electrostatic interaction, and chemical modification. Furthermore, the physiological roles of phase separation in cells, including molecules transport channel, gene expression and regulation, cellular division and differentiation, stress response, proteins refolding and degradation, cell connections, construction of skin barrier, and cell signals transmission, are highlighted. However, the disorder of phase separation leads to pathological condensates, which are associated with neurodegenerative diseases, tumors, and severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). This relationship is considered the potential target for developing corresponded drugs and therapy. Some drugs targeting phase separation have improved meaningful, such as tankyrase, lipoamide, oligonucleotides, elvitagravir, nilotinib, CVL218, PJ34. All in all, mystery phase separation provides a new viewpoint for researchers to explore cells, and is expected to solve many unknown phenomena in nature.

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mTOR Regulates Phase Separation of PGL Granules to Modulate Their Autophagic Degradation

Cited by 181 publications

References 32 publications

Germ Granules Functions are Memorized by Transgenerationally Inherited Small RNAs

Germ Granules Functions are Memorized by Transgenerationally Inherited Small RNAs

Liquid droplet germ granules require assembly and localized regulators for mRNA repression

Phase Separation: “The Master Key” to Deciphering the Physiological and Pathological Functions of Cells

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