Analysis of RNA associated with P granules in germ cells of C. elegans adults

Development ◽  
2001 ◽  
Vol 128 (8) ◽  
pp. 1287-1298 ◽  
Author(s):  
J.A. Schisa ◽  
J.N. Pitt ◽  
J.R. Priess
Keyword(s):  
Germ Cells ◽  
Rna Binding ◽  
Nuclear Surface ◽  
Binding Motifs ◽  
P Granules ◽  
C Elegans ◽  
Specific Mrnas ◽  
Tubulin Mrna ◽  
Protein Components ◽  
Cytoplasmic Structures

P granules are cytoplasmic structures of unknown function that are associated with germ nuclei in the C. elegans gonad, and are localized exclusively to germ cells, or germ cell precursors, throughout the life cycle. All the known protein components of P granules contain putative RNA-binding motifs, suggesting that RNA is involved in either the structure or function of the granules. However, no specific mRNAs have been identified within P granules in the gonad. We show here that P granules normally contain a low level of RNA, and describe conditions that increase this level. We present evidence that several, diverse mRNAs, including pos-1, mex-1, par-3, skn-1, nos-2 and gld-1 mRNA, are present at least transiently within P granules. In contrast, actin and tubulin mRNA and rRNA are either not present in P granules, or are present at relatively low levels. We show that pgl-1 and the glh (Vasa-related) gene family, which encode protein components of P granules, do not appear essential for RNA to concentrate in P granules; these proteins may instead function in events that are a prerequisite for RNAs to be transported efficiently from the nuclear surface.

The C. elegans MEX-1 protein is present in germline blastomeres and is a P granule component

Development ◽  
1997 ◽  
Vol 124 (3) ◽  
pp. 731-739 ◽  
Author(s):  
S. Guedes ◽  
J.R. Priess
Keyword(s):  
Germ Cells ◽  
Gene Products ◽  
Cytoplasmic Protein ◽  
Embryonic Cells ◽  
Nematode Caenorhabditis Elegans ◽  
P Granules ◽  
C Elegans ◽  
Cytoplasmic Structures ◽  
Fertilized Egg ◽  
Expression And Activity

In the nematode Caenorhabditis elegans, germ cells arise from early embryonic cells called germline blastomeres. Cytoplasmic structures called P granules are present in the fertilized egg and are segregated into each of the germline blastomeres during the first few cleavages of the embryo. Mutations in the maternally expressed gene mex-1 disrupt the segregation of P granules, prevent the formation of germ cells, and cause inappropriate patterns of somatic cell differentiation. We have cloned the mex-1 gene and determined the distribution pattern of the mex-1 gene products. The MEX-1 protein contains two copies of an unusual ‘finger’ domain also found in the PIE-1 protein of C. elegans. PIE-1 has been shown to be expressed in germline blastomeres, and is a component of P granules. We show here that MEX-1 also is present in germline blastomeres and is a P granule component, although MEX-1 is a cytoplasmic protein while PIE-1 is present in both the nucleus and cytoplasm. We further show that MEX-1 is required to restrict PIE-1 expression and activity to the germline blastomeres during the early embryonic cleavages.

scholarly journals Perinuclear P granules are the principal sites of mRNA export in adult C. elegans germ cells

Development ◽  
2010 ◽  
Vol 137 (8) ◽  
pp. 1305-1314 ◽  
Author(s):  
U. Sheth ◽  
J. Pitt ◽  
S. Dennis ◽  
J. R. Priess
Keyword(s):  
Germ Cells ◽  
Mrna Export ◽  
P Granules ◽  
C Elegans

scholarly journals Specification of the germline by Nanos-dependent down-regulation of the somatic synMuvB transcription factor LIN-15B

2017 ◽  
Author(s):  
Chih-Yung S. Lee ◽  
Tu Lu ◽  
Geraldine Seydoux
Keyword(s):  
Transcription Factor ◽  
Germ Cells ◽  
Rna Binding ◽  
Primordial Germ Cells ◽  
Somatic Cells ◽  
Rna Binding Protein ◽  
Dependent Manner ◽  
Embryonic Cells ◽  
C Elegans ◽  
Underlying Mechanisms

AbstractThe Nanos RNA-binding protein has been implicated in the specification of primordial germ cells (PGCs) in metazoans, but the underlying mechanisms remain poorly understood. We have profiled the transcriptome of PGCs lacking the nanos homologues nos-1 and nos-2 iC. elegans. nos-1nos-2 PGCs fail to silence hundreds of genes normally expressed in oocytes and somatic cells, a phenotype reminiscent of PGCs lacking the repressive PRC2 complex. The nos-1nos-2 phenotype depends on LIN-15B, a broadly expressed synMuvB class transcription factor known to antagonize PRC2 activity in somatic cells. LIN-15B is maternally-inherited by all embryonic cells and is down-regulated specifically in PGCs in a nos-1nos-2-dependent manner. Consistent with LIN-15B being a critical target of Nanos regulation, inactivation of maternal LIN-15B restores fertility to nos-1nos-2 mutants. These studies demonstrate a central role for Nanos in reprogramming the transcriptome of PGCs away from an oocyte/somatic fate by down-regulating an antagonist of PRC2 activity.

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 mRNA localization is linked to translation regulation in the Caenorhabditis elegans germ lineage

2020 ◽  
Author(s):  
Dylan M. Parker ◽  
Lindsay P. Winkenbach ◽  
Samuel P. Boyson ◽  
Matthew N. Saxton ◽  
Camryn Daidone ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Regulatory Control ◽  
Translational Repression ◽  
Translation Regulation ◽  
Germline Development ◽  
P Granules ◽  
C Elegans ◽  
Early Embryos

AbstractCaenorhabditis elegans early embryos generate cell-specific transcriptomes despite lacking active transcription. This presents an opportunity to study mechanisms of post-transcriptional regulatory control. In seeking the mechanisms behind this patterning, we discovered that some cell-specific mRNAs accumulate non-homogenously within cells, localizing to membranes, P granules (associated with progenitor germ cells in the P lineage), and P-bodies (associated with RNA processing). Transcripts differed in their dependence on 3’UTRs and RNA Binding Proteins, suggesting diverse regulatory mechanisms. Notably, we found strong but imperfect correlations between low translational status and P granule localization within the progenitor germ lineage. By uncoupling these, we untangled a long-standing question: Are mRNAs directed to P granules for translational repression or do they accumulate there as a downstream step? We found translational repression preceded P granule localization and could occur independent of it. Further, disruption of translation was sufficient to send homogenously distributed mRNAs to P granules. Overall, we show transcripts important for germline development are directed to P granules by translational repression, and this, in turn, directs their accumulation in the progenitor germ lineage where their repression can ultimately be relieved.SummaryMaternally loaded mRNAs localize non-homogeneously within C. elegans early embryos correlating with their translational status and lineage-specific fates.

scholarly journals An eIF4E-binding protein regulates katanin protein levels in C. elegans embryos

2009 ◽  
Vol 187 (1) ◽  
pp. 33-42 ◽  
Author(s):  
Wei Li ◽  
Leah R. DeBella ◽  
Tugba Guven-Ozkan ◽  
Rueyling Lin ◽  
Lesilee S. Rose
Keyword(s):  
Untranslated Region ◽  
Rna Binding ◽  
Binding Protein ◽  
Degradation Pathway ◽  
Translational Repression ◽  
Spindle Orientation ◽  
P Granules ◽  
C Elegans ◽  
Microtubule Severing ◽  
Protein Levels

In Caenorhabditis elegans, the MEI-1–katanin microtubule-severing complex is required for meiosis, but must be down-regulated during the transition to embryogenesis to prevent defects in mitosis. A cullin-dependent degradation pathway for MEI-1 protein has been well documented. In this paper, we report that translational repression may also play a role in MEI-1 down-regulation. Reduction of spn-2 function results in spindle orientation defects due to ectopic MEI-1 expression during embryonic mitosis. MEL-26, which is both required for MEI-1 degradation and is itself a target of the cullin degradation pathway, is present at normal levels in spn-2 mutant embryos, suggesting that the degradation pathway is functional. Cloning of spn-2 reveals that it encodes an eIF4E-binding protein that localizes to the cytoplasm and to ribonucleoprotein particles called P granules. SPN-2 binds to the RNA-binding protein OMA-1, which in turn binds to the mei-1 3′ untranslated region. Thus, our results suggest that SPN-2 functions as an eIF4E-binding protein to negatively regulate translation of mei-1.

scholarly journals Sam68 regulates translation of target mRNAs in male germ cells, necessary for mouse spermatogenesis

2009 ◽  
Vol 185 (2) ◽  
pp. 235-249 ◽  
Author(s):  
Maria Paola Paronetto ◽  
Valeria Messina ◽  
Enrica Bianchi ◽  
Marco Barchi ◽  
Gillian Vogel ◽  
...  
Keyword(s):  
Germ Cells ◽  
Male Fertility ◽  
Essential Role ◽  
Rna Binding ◽  
Mrna Translation ◽  
Male Gamete ◽  
Essential Requirement ◽  
Translational Machinery ◽  
Heterologous System ◽  
Specific Mrnas

Sam68 is a KH-type RNA-binding protein involved in several steps of RNA metabolism with potential implications in cell differentiation and cancer. However, its physiological roles are still poorly understood. Herein, we show that Sam68−/− male mice are infertile and display several defects in spermatogenesis, demonstrating an essential role for Sam68 in male fertility. Sam68−/− mice produce few spermatozoa, which display dramatic motility defects and are unable to fertilize eggs. Expression of a subset of messenger mRNAs (mRNAs) is affected in the testis of knockout mice. Interestingly, Sam68 is associated with polyadenylated mRNAs in the cytoplasm during the meiotic divisions and in round spermatids, when it interacts with the translational machinery. We show that Sam68 is required for polysomal recruitment of specific mRNAs and for accumulation of the corresponding proteins in germ cells and in a heterologous system. These observations demonstrate a novel role for Sam68 in mRNA translation and highlight its essential requirement for the development of a functional male gamete.

scholarly journals Transgenerational Epigenetic Inheritance Factors Localize to Spatially and Temporally Ordered Liquid Droplet Assemblages

2017 ◽  
Author(s):  
Gang Wan ◽  
Brandon D. Fields ◽  
George Spracklin ◽  
Carolyn Phillips ◽  
Scott Kennedy
Keyword(s):  
Rna Binding ◽  
Liquid Droplet ◽  
Epigenetic Inheritance ◽  
Liquid Droplets ◽  
P Granules ◽  
Transgenerational Epigenetic Inheritance ◽  
C Elegans ◽  
Regulatory Systems ◽  
Processing Pathways ◽  
Non Coding Rnas

AbstractEpigenetic information can be inherited for multiple generations (termed transgenerational epigenetic inheritance or TEI) 1,2. Non-coding RNAs have emerged as important mediators of TEI, although the mechanism(s) by which non-coding RNAs mediate TEI remains poorly understood. dsRNA-mediated gene silencing (RNAi) in C. elegans is a robust example of RNA-directed TEI3–5. To further our understanding of RNA-directed TEI, we conducted a genetic screen in C. elegans to identify genes required for RNAi inheritance. Our screen identified the conserved RNA helicase/Zn finger protein ZNFX-1 and the Argonaute protein WAGO-4. We find that WAGO-4 and ZNFX-1 act cooperatively in inheriting generations to maintain small interfering (si)RNA expression over generational time. ZNFX-1/ WAGO-4 localize to a liquid droplet organelle termed the P granule in early germline blastomeres. Later in development, ZNFX-1/WAGO-4 appear to separate from P granules to form independent foci that are adjacent to, yet remain distinct, from P granules. ZNFX-1/WAGO-4 labeled foci exhibit properties reminiscent of liquid droplets and we name these foci Z granules. In the adult germline, Z granules assemble into ordered tri-droplet assemblages with P granules and another germline droplet-like foci termed the Mutator foci. This work identifies a conserved RNA-binding protein that drives RNA-directed TEI in C. elegans, defines a new germline foci that we term the Z granule, demonstrates that liquid droplet formation is under developmental control, and shows that liquid droplets can assemble into spatially ordered multi-droplet structures. We speculate that temporal and spatial ordering of liquid droplets helps cells organize and coordinate the complex RNA processing pathways underlying gene regulatory systems, such as RNA-directed TEI.

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.

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