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1.
Nat Commun ; 15(1): 4127, 2024 May 15.
Article in English | MEDLINE | ID: mdl-38750080

ABSTRACT

Stress granules (SGs) are induced by various environmental stressors, resulting in their compositional and functional heterogeneity. SGs play a crucial role in the antiviral process, owing to their potent translational repressive effects and ability to trigger signal transduction; however, it is poorly understood how these antiviral SGs differ from SGs induced by other environmental stressors. Here we identify that TRIM25, a known driver of the ubiquitination-dependent antiviral innate immune response, is a potent and critical marker of the antiviral SGs. TRIM25 undergoes liquid-liquid phase separation (LLPS) and co-condenses with the SG core protein G3BP1 in a dsRNA-dependent manner. The co-condensation of TRIM25 and G3BP1 results in a significant enhancement of TRIM25's ubiquitination activity towards multiple antiviral proteins, which are mainly located in SGs. This co-condensation is critical in activating the RIG-I signaling pathway, thus restraining RNA virus infection. Our studies provide a conceptual framework for better understanding the heterogeneity of stress granule components and their response to distinct environmental stressors.


Subject(s)
DNA Helicases , Poly-ADP-Ribose Binding Proteins , RNA Helicases , RNA Recognition Motif Proteins , Signal Transduction , Stress Granules , Tripartite Motif Proteins , Ubiquitin-Protein Ligases , Ubiquitination , Humans , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , Tripartite Motif Proteins/metabolism , Tripartite Motif Proteins/genetics , RNA Recognition Motif Proteins/metabolism , RNA Recognition Motif Proteins/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Stress Granules/metabolism , RNA Helicases/metabolism , DNA Helicases/metabolism , DEAD Box Protein 58/metabolism , Transcription Factors/metabolism , Transcription Factors/genetics , Immunity, Innate , RNA, Double-Stranded/metabolism , HEK293 Cells , HeLa Cells , Cytoplasmic Granules/metabolism , RNA Virus Infections/virology , RNA Virus Infections/metabolism , RNA Virus Infections/immunology , Receptors, Immunologic/metabolism
2.
Front Immunol ; 15: 1358036, 2024.
Article in English | MEDLINE | ID: mdl-38690262

ABSTRACT

Background: It remains unclear whether BPIV3 infection leads to stress granules formation and whether G3BP1 plays a role in this process and in viral replication. This study aims to clarify the association between BPIV3 and stress granules, explore the effect of G3BP1 on BPIV3 replication, and provide significant insights into the mechanisms by which BPIV3 evades the host's antiviral immunity to support its own survival. Methods: Here, we use Immunofluorescence staining to observe the effect of BPIV3 infection on the assembly of stress granules. Meanwhile, the expression changes of eIF2α and G3BP1 were determined. Overexpression or siRNA silencing of intracellular G3BP1 levels was examined for its regulatory control of BPIV3 replication. Results: We identify that the BPIV3 infection elicited phosphorylation of the eIF2α protein. However, it did not induce the assembly of stress granules; rather, it inhibited the formation of stress granules and downregulated the expression of G3BP1. G3BP1 overexpression facilitated the formation of stress granules within cells and hindered viral replication, while G3BP1 knockdown enhanced BPIV3 expression. Conclusion: This study suggest that G3BP1 plays a crucial role in BPIV3 suppressing stress granule formation and viral replication.


Subject(s)
DNA Helicases , Poly-ADP-Ribose Binding Proteins , RNA Helicases , RNA Recognition Motif Proteins , Stress Granules , Virus Replication , Animals , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , RNA Recognition Motif Proteins/metabolism , DNA Helicases/metabolism , DNA Helicases/genetics , RNA Helicases/metabolism , RNA Helicases/genetics , Stress Granules/metabolism , Cattle , Eukaryotic Initiation Factor-2/metabolism , Respirovirus Infections/immunology , Respirovirus Infections/metabolism , Host-Pathogen Interactions/immunology , Phosphorylation , Cell Line , Cytoplasmic Granules/metabolism
3.
Sci Adv ; 10(18): eadg8771, 2024 May 03.
Article in English | MEDLINE | ID: mdl-38691600

ABSTRACT

To facilitate the interrogation of protein function at scale, we have developed high-throughput insertion of tags across the genome (HITAG). HITAG enables users to rapidly produce libraries of cells, each with a different protein of interest C-terminally tagged. HITAG is based on a modified strategy for performing Cas9-based targeted insertions, coupled with an improved approach for selecting properly tagged lines. Analysis of the resulting clones generated by HITAG reveals high tagging specificity, with most successful tagging events being indel free. Using HITAG, we fuse mCherry to a set of 167 stress granule-associated proteins and elucidate the features that drive a subset of proteins to strongly accumulate within these transient RNA-protein granules.


Subject(s)
Genetic Loci , Humans , CRISPR-Cas Systems , Proteins/genetics , Proteins/metabolism , High-Throughput Screening Assays/methods , Cytoplasmic Granules/metabolism , Cytoplasmic Granules/genetics
4.
Int Rev Neurobiol ; 176: 455-479, 2024.
Article in English | MEDLINE | ID: mdl-38802180

ABSTRACT

Amyotrophic lateral sclerosis (ALS) and related neurodegenerative diseases are characterised by dysfunction of a host of RNA-binding proteins (RBPs) and a severely disrupted RNA metabolism. Recently, RBP-harbouring phase-separated complexes, ribonucleoprotein (RNP) granules, have come into the limelight as "crucibles" of neuronal pathology in ALS. RNP granules are indispensable for the multitude of regulatory processes underlying cellular RNA metabolism and serve as critical organisers of cellular biochemistry. Neurons, highly specialised cells, heavily rely on RNP granules for efficient trafficking, signalling and stress responses. Multiple RNP granule components, primarily RBPs such as TDP-43 and FUS, are affected by ALS mutations. However, even in the absence of mutations, RBP proteinopathies represent pathophysiological hallmarks of ALS. Given the high local concentrations of RBPs and RNAs, their weakened or enhanced interactions within RNP granules disrupt their homeostasis. Thus, the physiological process of phase separation and RNP granule formation, vital for maintaining the high-functioning state of neuronal cells, becomes their Achilles heel. Here, we will review the recent literature on the causes and consequences of abnormal RNP granule functioning in ALS and related disorders. In particular, we will summarise the evidence for the network-level dysfunction of RNP granules in these conditions and discuss considerations for therapeutic interventions to target RBPs, RNP granules and their network as a whole.


Subject(s)
Amyotrophic Lateral Sclerosis , Cytoplasmic Granules , Ribonucleoproteins , Humans , Amyotrophic Lateral Sclerosis/metabolism , Amyotrophic Lateral Sclerosis/pathology , Ribonucleoproteins/metabolism , Animals , Cytoplasmic Granules/metabolism , Neurodegenerative Diseases/metabolism , Organelles/metabolism
6.
FEBS Lett ; 598(7): 774-786, 2024 Apr.
Article in English | MEDLINE | ID: mdl-38499396

ABSTRACT

Membraneless organelles are RNA-protein assemblies which have been implicated in post-transcriptional control. Germ cells form membraneless organelles referred to as germ granules, which contain conserved proteins including Tudor domain-containing scaffold polypeptides and their partner proteins that interact with Tudor domains. Here, we show that in Drosophila, different germ granule proteins associate with the multi-domain Tudor protein using different numbers of Tudor domains. Furthermore, these proteins compete for interaction with Tudor in vitro and, surprisingly, partition to distinct and poorly overlapping clusters in germ granules in vivo. This partition results in minimization of the competition. Our data suggest that Tudor forms structurally different configurations with different partner proteins which dictate different biophysical properties and phase separation parameters within the same granule.


Subject(s)
Drosophila Proteins , Drosophila , Animals , Cytoplasmic Granules/metabolism , Drosophila/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Germ Cell Ribonucleoprotein Granules , Germ Cells/metabolism
8.
Cell Rep ; 43(3): 113836, 2024 Mar 26.
Article in English | MEDLINE | ID: mdl-38421874

ABSTRACT

Endocrine cells employ regulated exocytosis of secretory granules to secrete hormones and neurotransmitters. Secretory granule exocytosis depends on spatiotemporal variables such as proximity to the plasma membrane and age, with newly generated granules being preferentially released. Despite recent advances, we lack a comprehensive view of the molecular composition of insulin granules and associated changes over their lifetime. Here, we report a strategy for the purification of insulin secretory granules of distinct age from insulinoma INS-1 cells. Tagging the granule-resident protein phogrin with a cleavable CLIP tag, we obtain intact fractions of age-distinct granules for proteomic and lipidomic analyses. We find that the lipid composition changes over time, along with the physical properties of the membrane, and that kinesin-1 heavy chain (KIF5b) as well as Ras-related protein 3a (RAB3a) associate preferentially with younger granules. Further, we identify the Rho GTPase-activating protein (ARHGAP1) as a cytosolic factor associated with insulin granules.


Subject(s)
Insulinoma , Pancreatic Neoplasms , Humans , Insulin/metabolism , Proteomics , Lipidomics , Insulinoma/metabolism , Pancreatic Neoplasms/metabolism , Exocytosis , Secretory Vesicles/metabolism , Cytoplasmic Granules/metabolism
9.
Nat Commun ; 15(1): 1524, 2024 Feb 19.
Article in English | MEDLINE | ID: mdl-38374028

ABSTRACT

Oligodendrocyte (OL) injury and subsequent loss is a pathologic hallmark of multiple sclerosis (MS). Stress granules (SGs) are membrane-less organelles containing mRNAs stalled in translation and considered as participants of the cellular response to stress. Here we show SGs in OLs in active and inactive areas of MS lesions as well as in normal-appearing white matter. In cultures of primary human adult brain derived OLs, metabolic stress conditions induce transient SG formation in these cells. Combining pro-inflammatory cytokines, which alone do not induce SG formation, with metabolic stress results in persistence of SGs. Unlike sodium arsenite, metabolic stress induced SG formation is not blocked by the integrated stress response inhibitor. Glycolytic inhibition also induces persistent SGs indicating the dependence of SG formation and disassembly on the energetic glycolytic properties of human OLs. We conclude that SG persistence in OLs in MS reflects their response to a combination of metabolic stress and pro-inflammatory conditions.


Subject(s)
Cytoplasmic Granules , Multiple Sclerosis , Humans , Cytoplasmic Granules/metabolism , Stress Granules , Oligodendroglia , Cytokines/metabolism , Stress, Physiological , Multiple Sclerosis/metabolism
10.
J Cell Biol ; 223(4)2024 Apr 01.
Article in English | MEDLINE | ID: mdl-38416111

ABSTRACT

Two mother centrioles in an animal cell are linked by intercentriolar fibers that have CROCC/rootletin as their main building block. Here, we investigated the regulatory role of intercentriolar/rootlet fibers in cilia assembly. The cilia formation rates were significantly reduced in the CEP250/C-NAP1 and CROCC/rootletin knockout (KO) cells, irrespective of the departure of the young mother centrioles from the basal bodies. In addition, centriolar satellites were dispersed throughout the cytoplasm in the CEP250 and CROCC KO cells. We observed that PCM1 directly binds to CROCC. Their interaction is critical not only for the accumulation of centriolar satellites near the centrosomes/basal bodies but also for cilia formation. Finally, we observed that the centriolar satellite proteins are localized at the intercentriolar/rootlet fibers in the kidney epithelial cells. Based on these findings, we propose that the intercentriolar/rootlet fibers function as docking sites for centriolar satellites near the centrosomes/basal bodies and facilitate the cilia assembly process.


Subject(s)
Centrioles , Cilia , Basal Bodies , Centrioles/genetics , Centrosome , Cytoplasmic Granules , Humans , Epithelial Cells/cytology
11.
Microb Cell Fact ; 23(1): 52, 2024 Feb 15.
Article in English | MEDLINE | ID: mdl-38360657

ABSTRACT

BACKGROUND: Among the polyhydroxyalkanoate (PHA), poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxyhexanoate] [P(3HB-co-3HHx)] is reported to closely resemble polypropylene and low-density polyethylene. Studies have shown that PHA synthase (PhaC) from mangrove soil (PhaCBP-M-CPF4) is an efficient PhaC for P(3HB-co-3HHx) production and N-termini of PhaCs influence its substrate specificity, dimerization, granule morphology, and molecular weights of PHA produced. This study aims to further improve PhaCBP-M-CPF4 through N-terminal truncation. RESULTS: The N-terminal truncated mutants of PhaCBP-M-CPF4 were constructed based on the information of the predicted secondary and tertiary structures using PSIPRED server and AlphaFold2 program, respectively. The N-terminal truncated PhaCBP-M-CPF4 mutants were evaluated in C. necator mutant PHB-4 based on the cell dry weight, PHA content, 3HHx molar composition, molecular weights, and granule morphology of the PHA granules. The results showed that most transformants harbouring the N-terminal truncated PhaCBP-M-CPF4 showed a reduction in PHA content and cell dry weight except for PhaCBP-M-CPF4 G8. PhaCBP-M-CPF4 G8 and A27 showed an improved weight-average molecular weight (Mw) of PHA produced due to lower expression of the truncated PhaCBP-M-CPF4. Transformants harbouring PhaCBP-M-CPF4 G8, A27, and T74 showed a reduction in the number of granules. PhaCBP-M-CPF4 G8 produced higher Mw PHA in mostly single larger PHA granules with comparable production as the full-length PhaCBP-M-CPF4. CONCLUSION: This research showed that N-terminal truncation had effects on PHA accumulation, substrate specificity, Mw, and granule morphology. This study also showed that N-terminal truncation of the amino acids that did not adopt any secondary structure can be an alternative to improve PhaCs for the production of PHA with higher Mw in mostly single larger granules.


Subject(s)
Cupriavidus necator , Polyhydroxyalkanoates , Polyhydroxyalkanoates/metabolism , 3-Hydroxybutyric Acid , Caproates/metabolism , Hydroxybutyrates/metabolism , Acyltransferases/genetics , Acyltransferases/metabolism , Cytoplasmic Granules , Cupriavidus necator/genetics , Cupriavidus necator/metabolism
12.
Nucleic Acids Res ; 52(9): 5356-5375, 2024 May 22.
Article in English | MEDLINE | ID: mdl-38366783

ABSTRACT

Stress granules (SGs) are cytoplasmic assemblies formed under various stress conditions as a consequence of translation arrest. SGs contain RNA-binding proteins, ribosomal subunits and messenger RNAs (mRNAs). It is well known that mRNAs contribute to SG formation; however, the connection between SG assembly and nuclear processes that involve mRNAs is not well established. Here, we examine the effects of inhibiting mRNA transcription, splicing and export on the assembly of SGs and the related cytoplasmic P body (PB). We demonstrate that inhibition of mRNA transcription, splicing and export reduces the formation of canonical SGs in a eukaryotic initiation factor 2α phosphorylation-independent manner, and alters PB size and quantity. We find that the splicing inhibitor madrasin promotes the assembly of stress-like granules. We show that the addition of synthetic mRNAs directly to the cytoplasm is sufficient for SG assembly, and that the assembly of these SGs requires the activation of stress-associated protein synthesis pathways. Moreover, we show that adding an excess of mRNA to cells that do not have active splicing, and therefore have low levels of cytoplasmic mRNAs, promotes SG formation under stress conditions. These findings emphasize the importance of the cytoplasmic abundance of newly transcribed mRNAs in the assembly of SGs.


Subject(s)
Cell Nucleus , Cytoplasmic Granules , RNA Splicing , RNA, Messenger , Cytoplasmic Granules/metabolism , RNA, Messenger/metabolism , RNA, Messenger/genetics , Humans , Cell Nucleus/metabolism , Cell Nucleus/genetics , Stress Granules/metabolism , Transcription, Genetic , Cytoplasm/metabolism , HeLa Cells , Eukaryotic Initiation Factor-2/metabolism , Phosphorylation
14.
Nat Commun ; 15(1): 407, 2024 Jan 09.
Article in English | MEDLINE | ID: mdl-38195629

ABSTRACT

T-cell cytotoxic function relies on the cooperation between the highly specific but poorly adhesive T-cell receptor (TCR) and the integrin LFA-1. How LFA-1-mediated adhesion may scale with TCR stimulation strength is ill-defined. Here, we show that LFA-1 conformation activation scales with TCR stimulation to calibrate human T-cell cytotoxicity. Super-resolution microscopy analysis reveals that >1000 LFA-1 nanoclusters provide a discretized platform at the immunological synapse to translate TCR engagement and density of the LFA-1 ligand ICAM-1 into graded adhesion. Indeed, the number of high-affinity conformation LFA-1 nanoclusters increases as a function of TCR triggering strength. Blockade of LFA-1 conformational activation impairs adhesion to target cells and killing. However, it occurs at a lower TCR stimulation threshold than lytic granule exocytosis implying that it licenses, rather than directly controls, the killing decision. We conclude that the organization of LFA-1 into nanoclusters provides a calibrated system to adjust T-cell killing to the antigen stimulation strength.


Subject(s)
Antineoplastic Agents , T-Lymphocytes , Humans , Cytoplasmic Granules , Lymphocyte Function-Associated Antigen-1 , Receptors, Antigen, T-Cell , CD11a Antigen/metabolism
15.
Semin Cell Dev Biol ; 159-160: 10-26, 2024.
Article in English | MEDLINE | ID: mdl-38278052

ABSTRACT

Stress granules and P-bodies are conserved cytoplasmic biomolecular condensates whose assembly and composition are well documented, but whose clearance mechanisms remain controversial or poorly described. Such understanding could provide new insight into how cells regulate biomolecular condensate formation and function, and identify therapeutic strategies in disease states where aberrant persistence of stress granules in particular is implicated. Here, I review and compare the contributions of chaperones, the cytoskeleton, post-translational modifications, RNA helicases, granulophagy and the proteasome to stress granule and P-body clearance. Additionally, I highlight the potentially vital role of RNA regulation, cellular energy, and changes in the interaction networks of stress granules and P-bodies as means of eliciting clearance. Finally, I discuss evidence for interplay of distinct clearance mechanisms, suggest future experimental directions, and suggest a simple working model of stress granule clearance.


Subject(s)
Processing Bodies , Stress Granules , Cytoplasmic Granules , RNA Helicases , Cytoplasm
16.
Nucleic Acids Res ; 52(6): 3310-3326, 2024 Apr 12.
Article in English | MEDLINE | ID: mdl-38165001

ABSTRACT

L1TD1 is a cytoplasmic RNA-binding protein specifically expressed in pluripotent stem cells and, unlike its mouse ortholog, is essential for the maintenance of stemness in human cells. Although L1TD1 is the only known protein-coding gene domesticated from a LINE-1 (L1) retroelement, the functional legacy of its ancestral protein, ORF1p of L1, and how it is manifested in L1TD1 are still unknown. Here, we determined RNAs associated with L1TD1 and found that, like ORF1p, L1TD1 binds L1 RNAs and localizes to high-density ribonucleoprotein (RNP) condensates. Unexpectedly, L1TD1 enhanced the translation of a subset of mRNAs enriched in the condensates. L1TD1 depletion promoted the formation of stress granules in embryonic stem cells. In HeLa cells, ectopically expressed L1TD1 facilitated the dissolution of stress granules and granules formed by pathological mutations of TDP-43 and FUS. The glutamate-rich domain and the ORF1-homology domain of L1TD1 facilitated dispersal of the RNPs and induced autophagy, respectively. These results provide insights into how L1TD1 regulates gene expression in pluripotent stem cells. We propose that the ability of L1TD1 to dissolve stress granules may provide novel opportunities for treatment of neurodegenerative diseases caused by disturbed stress granule dynamics.


Subject(s)
Embryonic Stem Cells , RNA-Binding Proteins , Ribonucleoproteins , Animals , Humans , Mice , Cytoplasmic Granules/metabolism , Embryonic Stem Cells/cytology , Embryonic Stem Cells/metabolism , HeLa Cells , Ribonucleoproteins/genetics , Ribonucleoproteins/metabolism , RNA/metabolism , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism
17.
Biochem Biophys Res Commun ; 697: 149497, 2024 Feb 19.
Article in English | MEDLINE | ID: mdl-38262290

ABSTRACT

Stress granule (SG) is a temporary cellular structure that plays a crucial role in the regulation of mRNA and protein sequestration during various cellular stress conditions. SG enables cells to cope with stress more effectively, conserving vital energy and resources. Focusing on the NTF2-like domain of G3BP1, a key protein in SG dynamics, we explore to identify and characterize novel small molecules involved in SG modulation without external stressors. Through in silico molecular docking approach to simulate the interaction between various compounds and the NTF2-like domain of G3BP1, we identified three compounds as potential candidates that could bind to the NTF2-like domain of G3BP1. Subsequent immunofluorescence experiments demonstrated that these compounds induce the formation of SG-like, G3BP1-positive granules. Importantly, the granule formation by these compounds occurs independent from the phosphorylation of eIF2α, a common mechanism in SG formation, suggesting that it might offer a new strategy for influencing SG dynamics implicated in various diseases.


Subject(s)
DNA Helicases , RNA Helicases , DNA Helicases/metabolism , RNA Helicases/metabolism , RNA Recognition Motif Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Molecular Docking Simulation , Cytoplasmic Granules/metabolism
18.
Geriatr Gerontol Int ; 24 Suppl 1: 7-14, 2024 Mar.
Article in English | MEDLINE | ID: mdl-37726158

ABSTRACT

Living organisms experience a range of stresses. To cope effectively with these stresses, eukaryotic cells have evolved a sophisticated mechanism involving the formation of stress granules (SGs), which play a crucial role in protecting various types of RNA species under stress, such as mRNAs and long non-coding RNAs (lncRNAs). SGs are non-membranous cytoplasmic ribonucleoprotein (RNP) granules, and the RNAs they contain are translationally stalled. Importantly, SGs have been thought to contribute to the pathophysiology of neurodegenerative diseases, including Alzheimer's disease (AD). SGs also contain multiple RNA-binding proteins (RBPs), several of which have been implicated in AD progression. SGs are transient structures that dissipate after stress relief. However, the chronic stresses associated with aging lead to the persistent formation of SGs and subsequently to solid-like pathological SGs, which could impair cellular RNA metabolism and also act as a nidus for the aberrant aggregation of AD-associated proteins. In this paper, we provide a comprehensive summary of the physical basis of SG-enriched RNAs and SG-resident RBPs. We then review the characteristics of AD-associated gene transcripts and their similarity to the SG-enriched RNAs. Furthermore, we summarize and discuss the functional implications of SGs in neuronal RNA metabolism and the aberrant aggregation of AD-associated proteins mediated by SG-resident RBPs in the context of AD pathogenesis. Geriatr Gerontol Int 2024; 24: 7-14.


Subject(s)
Alzheimer Disease , RNA , Humans , RNA/genetics , RNA/metabolism , Alzheimer Disease/pathology , Stress Granules , Cytoplasmic Granules/genetics , Cytoplasmic Granules/metabolism , Cytoplasmic Granules/pathology , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolism
19.
Zygote ; 32(1): 28-37, 2024 Feb.
Article in English | MEDLINE | ID: mdl-38047350

ABSTRACT

Oocyte cytoplasmic evaluation is based on homogeneity and granular appearance. Our study investigated if a granular cytoplasm, highly heterogeneous, would affect oocyte competence in bovine. In two experiments, bovine cumulus-oocyte complexes (COCs) with homogeneous cytoplasm (control, CC) and granulated cytoplasm (granular, GC) were selected from a regular pool of COCs. Experiment 1 was performed with slaughterhouse ovaries, and Experiment 2 was carried out in Girolando COCs obtained from ovum pick-up. Granular oocytes had higher caspase 3 levels (66.17 ± 11.61 vs 172.08 ± 16.95, P < 0.01) and similar GAP junction activity (5.64 ± 0.45 vs 6.29 ± 0.29). ZAR1 relative mRNA amount was lower in granular oocytes (178.27 ± 151.63 vs 0.89 ± 0.89, P = 0.01) and no effect was detected for MATER, PPP2R1A, ENY2, IGF2R, and BMP15 genes. Despite molecular differences, no detrimental effect was detected on oocyte competence in GC oocytes. Cleavage (Experiment 1: 59.52 ± 7.21% vs 59.79 ± 6.10% and Experiment 2: 68.88 ± 4.82 vs 74.41 ± 5.89%) and blastocyst (Experiment 1: 29.28 ± 4.14% vs 23.15 ± 2.96% and Experiment 2: 21.11 ± 3.28% vs 21.02 ± 6.08%) rates were similar between CC and GC (Experiments 1 and 2, respectively). Post-transfer embryo development revealed that pregnancy (CC: 24.27 ± 9.70% vs GC: 26.31 ± 7.23%) and calving (23.68% vs 33.33%) rates and fetal growth were not affected by the presence of cytoplasmic granules. Our results demonstrated that oocytes with granular cytoplasm present equivalent efficiency for IVF and calf production compared with homogenous cytoplasm oocytes. This could be observed through similar cleavage, blastocyst rates, and fetal growth development. In addition to differences in oocyte gene expression related to oocyte quality, it seems not to affect oocyte developmental competence.


Subject(s)
Embryonic Development , Oocytes , Pregnancy , Female , Animals , Cattle , Oocytes/metabolism , Oogenesis , Fetal Development , Cytoplasmic Granules , In Vitro Oocyte Maturation Techniques/methods
20.
Semin Cell Dev Biol ; 156: 160-166, 2024 03 15.
Article in English | MEDLINE | ID: mdl-36202692

ABSTRACT

Stress granules (SGs), structurally dynamic, optically resolvable, macromolecular assemblies of mRNAs, RNA binding proteins (RBPs), translation factors, ribosomal subunits, as well as other interacting proteins, assemble in response to cell stress conditions that elicit phosphorylation of eukaryotic initiation factor 2α (eIF2α) and consequently, the inactivation of translation initiation. SG biology is conserved throughout eukaryotes and has recently been linked to the pathological sequelae of neurodegenerative disorders, cancer biology, and viral infection. Substantial insights into mechanisms of SG biogenesis, and more broadly the phenomenon of biological liquid-liquid phase separation (LLPS), have been aided by detailed proteomic and transcriptomic studies as well as in vitro reconstitution approaches. A particularly interesting and largely unexplored element of SG biology is the cell biological context of SG biogenesis, including its subcellular organization and more recently, evidence that the endoplasmic reticulum (ER) membrane may serve important functions in RNA granule biology generally and SG biogenesis specifically. A central role for the ER in SG biogenesis is discussed and a hypothesis linking SG formation on the ER to the trafficking, localization and de novo translation of newly exported mRNAs is presented.


Subject(s)
Proteomics , Stress Granules , Cytoplasmic Granules , Endoplasmic Reticulum/metabolism , RNA-Binding Proteins/metabolism , RNA, Messenger/metabolism
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