scholarly journals Germline maintenance through the multifaceted activities of GLH/Vasa in Caenorhabditis elegans P granules

2019 ◽  
Author(s):  
Elisabeth A. Marnik ◽  
J. Heath Fuqua ◽  
Catherine S. Sharp ◽  
Jesse D. Rochester ◽  
Emily L. Xu ◽  
...  
Keyword(s):  
Small Rna ◽  
Nuclear Periphery ◽  
Protein Translation ◽  
26S Proteasome ◽  
Mass Spectrometry Analysis ◽  
List Type ◽  
Helicase Activity ◽  
Critical Component ◽  
P Granules ◽  
C Elegans

SUMMARYVasa is a highly conserved member of the ATP-dependent DEAD box helicase family, a multipotency factor, and a critical component for the specification and maintenance of the germline. Its homologs have been shown to regulate translation, small RNA amplification, and serve as a molecular solvent for single-stranded RNA; however, the function of Vasa’s defining domains and what they interact with are unclear. To address this, 28 mutant alleles of the C. elegans Vasa homolog GLH-1 were generated in conserved motifs. Mutations in the flanking and helicase domains show that GLH-1 retains its association with P granules through its helicase activity and not through static interactions with other P-granule proteins. Changes outside of these domains retain GLH-1 in P granules but still compromise fertility, and removal of glycine-rich repeats progressively diminish P-granule wetting-like interactions at the nuclear periphery. A mutation that facilitates Vasa aggregation was previously leveraged in insects and mammals to identify the transient association of Vasa with piRNA amplifying Argonautes. This same mutation in GLH-1 also stimulates aggregation and association with Argonautes, suggesting that the transient amplifying complex is evolutionarily conserved even though the method of piRNA amplification in C. elegans is not. Mass spectrometry analysis of proteins that co-immunoprecipitate with wild type and mutant GLH-1 reveal an affinity for all three PCI (26S Proteasome Lid, COP9, eIF3) scaffolding complexes, which regulate protein turnover and translation, and a possible aversion for ribosomes and the 26S proteasome core. These results suggest that phase-separated P granules compartmentalize the cytoplasm to exclude large protein assemblies and emphasize the role of Vasa homologs in maintaining proteostasis. GRAPHICAL ABSTRACTHIGHLIGHTSGLH-1/Vasa helicase activity is required for germ granule association and the flanking domain is critical component of this helicase activity.GLH-1 and GLH-2 glycine-rich FG-repeats increase the coverage or wetting-like properties of germ granules at the nuclear periphery.Locked GLH-1 helicase domains increase association with Argonaute proteins, resembling small RNA transient amplifying complexes observed in insects and mammals.GLH-1 has an affinity for all three PCI (26S Proteasome Lid, COP9, eIF3) scaffolding complexes, emphasizing a role in protein translation and turnover.

scholarly journals P granules extend the nuclear pore complex environment in the C. elegans germ line

2011 ◽  
Vol 192 (6) ◽  
pp. 939-948 ◽  
Author(s):  
Dustin L. Updike ◽  
Stephanie J. Hachey ◽  
Jeremy Kreher ◽  
Susan Strome
Keyword(s):  
Nuclear Pore Complex ◽  
Germ Plasm ◽  
Germ Line ◽  
Hydrophobic Interactions ◽  
Nuclear Periphery ◽  
Size Exclusion ◽  
Nuclear Pore ◽  
Complex Environment ◽  
P Granules ◽  
C Elegans

The immortal and totipotent properties of the germ line depend on determinants within the germ plasm. A common characteristic of germ plasm across phyla is the presence of germ granules, including P granules in Caenorhabditis elegans, which are typically associated with the nuclear periphery. In C. elegans, nuclear pore complex (NPC)–like FG repeat domains are found in the VASA-related P-granule proteins GLH-1, GLH-2, and GLH-4 and other P-granule components. We demonstrate that P granules, like NPCs, are held together by weak hydrophobic interactions and establish a size-exclusion barrier. Our analysis of intestine-expressed proteins revealed that GLH-1 and its FG domain are not sufficient to form granules, but require factors like PGL-1 to nucleate the localized concentration of GLH proteins. GLH-1 is necessary but not sufficient for the perinuclear location of granules in the intestine. Our results suggest that P granules extend the NPC environment in the germ line and provide insights into the roles of the PGL and GLH family proteins.

scholarly journals Germ Granules Functions are Memorized by Transgenerationally Inherited Small RNAs

2019 ◽  
Author(s):  
Itamar Lev ◽  
Itai Antoine Toker ◽  
Yael Mor ◽  
Anat Nitzan ◽  
Guy Weintraub ◽  
...  
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Early Embryo ◽  
Small Rna Sequencing ◽  
Wild Type ◽  
P Granules ◽  
Multiple Generations ◽  
C Elegans ◽  
The Core ◽  
Germ Granules

AbstractInC. elegansnematodes, 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 inpptr-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 ancestralmeg-3/4alleles 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 frommeg-3/4male 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 thathrde-1;meg-3/4triple mutants inherit RNAi, althoughhrde-1was 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.

scholarly journals Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Ian F Price ◽  
Hannah L Hertz ◽  
Benjamin Pastore ◽  
Jillian Wagner ◽  
Wen Tang
Keyword(s):  
Germ Line ◽  
Nuclear Periphery ◽  
P Granules ◽  
C Elegans ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Germ Granules ◽  
Protein Components ◽  
Labeling Method ◽  
Disordered Regions

The germ line produces gametes that transmit genetic and epigenetic information to the next generation. Maintenance of germ cells and development of gametes require germ granules-well-conserved membraneless and RNA-rich organelles. The composition of germ granules is elusive owing to their dynamic nature and their exclusive expression in the germ line. Using C. elegans germ granule, called P granule, as a model system, we employed a proximity-based labeling method in combination with mass spectrometry to comprehensively define its protein components. This set of experiments identified over 200 proteins, many of which contain intrinsically disordered regions. An RNAi-based screen identified factors that are essential for P granule assembly, notably EGGD-1 and EGGD-2, two putative LOTUS-domain proteins. Loss of eggd-1 and eggd-2 results in separation of P granules from the nuclear envelope, germline atrophy and reduced fertility. We show that intrinsically disordered regions of EGGD-1 are required to anchor EGGD-1 to the nuclear periphery while its LOTUS domains are required to promote perinuclear localization of P granules. Together, our work expands the repertoire of P granule constituents and provides new insights into the role of LOTUS-domain proteins in germ granule organization.

scholarly journals Proximity labeling identifies LOTUS domain proteins that promote the formation of perinuclear germ granules in C. elegans

2021 ◽  
Author(s):  
Wen Tang ◽  
Ian F. Price ◽  
Hannah L. Hertz ◽  
Benjamin Pastore ◽  
Jillian Wagner
Keyword(s):  
Nuclear Periphery ◽  
P Granules ◽  
C Elegans ◽  
Intrinsically Disordered ◽  
Intrinsically Disordered Regions ◽  
Germ Granules ◽  
Protein Components ◽  
Labeling Method ◽  
Disordered Regions

The germline produces gametes that transmit genetic and epigenetic information to the next generation. Maintenance of germ cells and development of gametes require germ granules—well-conserved membraneless and RNA-rich organelles. The composition of germ granules is elusive owing to their dynamic nature and their exclusive expression in the germline. Using C. elegans germ granule, called P granule, as a model system, we employed a proximity-based labeling method in combination with mass spectrometry to comprehensively define its protein components. This set of experiments identified over 200 proteins, many of which contain intrinsically disordered regions. An RNAi-based screen identified factors that are essential for P granule assembly, notably EGGD-1 and EGGD-2, two previously uncharacterized LOTUS-domain proteins. Loss of eggd-1 and eggd-2 results in separation of P granules from nuclear envelope, germline atrophy and reduced fertility. We show that intrinsically disordered regions of EGGD-1 are required to anchor EGGD-1 to the nuclear periphery while its LOTUS domains are required to promote perinuclear localization of P granules. Together, our work expands the repertoire of P granule constituents and provides new insights into the role of LOTUS-domain proteins in germ granule organization.

scholarly journals RNA-silencing induces target gene relocalization toward a specialized nuage domain

2021 ◽  
Author(s):  
Yuchen Yang ◽  
David Grunwald ◽  
James R. Priess ◽  
Craig C. Mello
Keyword(s):  
Small Rna ◽  
Rna Silencing ◽  
Target Gene ◽  
Developmentally Regulated ◽  
Rna Sequences ◽  
P Granules ◽  
C Elegans ◽  
Heterochromatin Silencing ◽  
Rna Pathways ◽  
Target Rna

Argonaute small RNA pathways maintain genome integrity and fertility by enforcing the transgenerational silencing of transposons as well as many developmentally regulated germline genes 1. To propagate silencing, Argonaute pathways coordinate heterochromatin silencing with cycles of small RNA amplification 2. In animal germlines, mRNA surveillance is thought to occur within cytoplasmic perinuclear domains called nuage 3. In C. elegans 20-50 nuage droplets called P granules surround each pachytene germline nucleus. P granules are known to host many of the Argonaute small RNA systems that carry out transcriptome surveillance, but what if any specific roles P granules might play in Argonaute silencing have remained mysterious. Here we show that RNAi triggers the expansion of a unique P granule, which accumulates large amounts of the target RNA. As transcriptional silencing ensues, both alleles of the target gene relocate near the inner nuclear membrane (INM) directly adjacent this enlarged P granule. Similarly, during piRNA-mediated silencing, both alleles of a target gene reside adjacent to a P granule containing target RNA sequences. In an Argonaute mutant defective in piRNA silencing, the target RNA is released from nuage, and the target alleles dissociate from each other and from the INM. Our findings suggest that transcriptome-surveillance tasks are sub-divided between nuage domains that become specialized to coordinate small RNA silencing signals to their heterochromatin targets.

scholarly journals C. elegans germ granules require both assembly and localized regulators for mRNA repression

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Scott Takeo Aoki ◽  
Tina R. Lynch ◽  
Sarah L. Crittenden ◽  
Craig A. Bingman ◽  
Marvin Wickens ◽  
...  
Keyword(s):  
Liquid Droplet ◽  
Scaffolding Protein ◽  
P Granules ◽  
C Elegans ◽  
Cytoplasmic Rna ◽  
And Function ◽  
Rnp Granules ◽  
Key Residues ◽  
Reporter Mrna

AbstractCytoplasmic RNA–protein (RNP) granules have diverse biophysical properties, from liquid to solid, and play enigmatic roles in RNA metabolism. Nematode P granules are paradigmatic liquid droplet granules and central to germ cell development. Here we analyze a key P granule scaffolding protein, PGL-1, to investigate the functional relationship between P granule assembly and function. Using a protein–RNA tethering assay, we find that reporter mRNA expression is repressed when recruited to PGL-1. We determine the crystal structure of the PGL-1 N-terminal region to 1.5 Å, discover its dimerization, and identify key residues at the dimer interface. Mutations of those interface residues prevent P granule assembly in vivo, de-repress PGL-1 tethered mRNA, and reduce fertility. Therefore, PGL-1 dimerization lies at the heart of both P granule assembly and function. Finally, we identify the P granule-associated Argonaute WAGO-1 as crucial for repression of PGL-1 tethered mRNA. We conclude that P granule function requires both assembly and localized regulators.

scholarly journals Re-Discovery of Giardiavirus: Genomic and Functional Analysis of Viruses from Giardia duodenalis Isolates

Biomedicines ◽  
2021 ◽  
Vol 9 (6) ◽  
pp. 654
Author(s):  
Gianluca Marucci ◽  
Ilaria Zullino ◽  
Lucia Bertuccini ◽  
Serena Camerini ◽  
Serena Cecchetti ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Diarrheal Disease ◽  
Current Knowledge ◽  
Biological Significance ◽  
Giardia Duodenalis ◽  
Protozoan Parasite ◽  
Protein Translation ◽  
Mass Spectrometry Analysis ◽  
Animal Origin ◽  
Spectrometry Analysis

Giardiasis, caused by the protozoan parasite Giardia duodenalis, is an intestinal diarrheal disease affecting almost one billion people worldwide. A small endosymbiotic dsRNA viruses, G. lamblia virus (GLV), genus Giardiavirus, family Totiviridae, might inhabit human and animal isolates of G. duodenalis. Three GLV genomes have been sequenced so far, and only one was intensively studied; moreover, a positive correlation between GLV and parasite virulence is yet to be proved. To understand the biological significance of GLV infection in Giardia, the characterization of several GLV strains from naturally infected G. duodenalis isolates is necessary. Here we report high-throughput sequencing of four GLVs strains, from Giardia isolates of human and animal origin. We also report on a new, unclassified viral sequence (designed GdRV-2), unrelated to Giardiavirus, encoding and expressing for a single large protein with an RdRp domain homologous to Totiviridae and Botybirnaviridae. The result of our sequencing and proteomic analyses challenge the current knowledge on GLV and strongly suggest that viral capsid protein translation unusually starts with a proline and that translation of the RNA-dependent RNA polymerase (RdRp) occurs via a +1/−2 ribosomal frameshift mechanism. Nucleotide polymorphism, confirmed by mass-spectrometry analysis, was also observed among and between GLV strains. Phylogenetic analysis indicated the occurrence of at least two GLV subtypes which display different phenotypes and transmissibility in experimental infections of a GLV naïve Giardia isolate.

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