Epstein-Barr Virus BGLF2 commandeers RISC to interfere with cellular miRNA function

The Epstein-Barr virus (EBV) BGLF2 protein is a tegument protein with multiple effects on the cellular environment, including induction of SUMOylation of cellular proteins. Using affinity-purification coupled to mass-spectrometry, we identified the miRNA-Induced Silencing Complex (RISC), essential for miRNA function, as a top interactor of BGLF2. We confirmed BGLF2 interaction with the Ago2 and TNRC6 components of RISC in multiple cell lines and their co-localization in cytoplasmic bodies that also contain the stress granule marker G3BP1. In addition, BGLF2 expression led to the loss of processing bodies in multiple cell types, suggesting disruption of RISC function in mRNA regulation. Consistent with this observation, BGLF2 disrupted Ago2 association with multiple miRNAs. Using let-7 miRNAs as a model, we tested the hypothesis that BGLF2 interfered with the function of RISC in miRNA-mediated mRNA silencing. Using multiple reporter constructs with 3’UTRs containing let-7a regulated sites, we showed that BGLF2 inhibited let-7a miRNA activity dependent on these 3’UTRs, including those from SUMO transcripts which are known to be regulated by let-7 miRNAs. In keeping with these results, we showed that BGLF2 increased the cellular level of unconjugated SUMO proteins without affecting the level of SUMO transcripts. Such an increase in free SUMO is known to drive SUMOylation and would account for the effect of BGLF2 in inducing SUMOylation. We further showed that BGLF2 expression inhibited the loading of let-7 miRNAs into Ago2 proteins, and conversely, that lytic infection with EBV lacking BGLF2 resulted in increased interaction of let-7a and SUMO transcripts with Ago2, relative to WT EBV infection. Therefore, we have identified a novel role for BGLF2 as a miRNA regulator and shown that one outcome of this activity is the dysregulation of SUMO transcripts that leads to increased levels of free SUMO proteins and SUMOylation.

Muscle-Related Plectinopathies

Plectin is a giant cytoskeletal crosslinker and intermediate filament stabilizing protein. Mutations in the human plectin gene (PLEC) cause several rare diseases that are grouped under the term plectinopathies. The most common disorder is autosomal recessive disease epidermolysis bullosa simplex with muscular dystrophy (EBS-MD), which is characterized by skin blistering and progressive muscle weakness. Besides EBS-MD, PLEC mutations lead to EBS with nail dystrophy, EBS-MD with a myasthenic syndrome, EBS with pyloric atresia, limb-girdle muscular dystrophy type R17, or EBS-Ogna. In this review, we focus on the clinical and pathological manifestations caused by PLEC mutations on skeletal and cardiac muscle. Skeletal muscle biopsies from EBS-MD patients and plectin-deficient mice revealed severe dystrophic features with variation in fiber size, degenerative myofibrillar changes, mitochondrial alterations, and pathological desmin-positive protein aggregates. Ultrastructurally, PLEC mutations lead to a disorganization of myofibrils and sarcomeres, Z- and I-band alterations, autophagic vacuoles and cytoplasmic bodies, and misplaced and degenerating mitochondria. We also summarize a variety of genetically manipulated mouse and cell models, which are either plectin-deficient or that specifically lack a skeletal muscle-expressed plectin isoform. These models are powerful tools to study functional and molecular consequences of PLEC defects and their downstream effects on the skeletal muscle organization.

Correlated cryogenic fluorescence microscopy and electron cryo-tomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo

Members of the tripartite motif (TRIM) protein family have been shown to assemble into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5α, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy, combined with electron cryo-tomography, to intact mammalian cells expressing YFP-rhTRIM5α and found the presence of hexagonal nets whose arm lengths were similar to those of the hexagonal nets formed by purified TRIM5α in vitro. We also observed YFP-rhTRIM5α within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within double-membraned vesicles (autophagosomes), and sequestosomes within multivesicular autophagic vacuoles (amphisomes or autolysosomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data 1) support recent reports that TRIM5α can form both well-organized signaling complexes and nonsignaling aggregates, 2) offer images of the macroautophagy pathway in a near-native state, and 3) reveal that vaults arrive early in macroautophagy.

Short Tandem Repeat-Enriched Architectural RNAs in Nuclear Bodies: Functions and Associated Diseases

Nuclear bodies are membraneless, phase-separated compartments that concentrate specific proteins and RNAs in the nucleus. They are believed to serve as sites for the modification, sequestration, and storage of specific factors, and to act as organizational hubs of chromatin structure to control gene expression and cellular function. Architectural (arc) RNA, a class of long noncoding RNA (lncRNA), plays essential roles in the formation of nuclear bodies. Herein, we focus on specific arcRNAs containing short tandem repeat-enriched sequences and introduce their biological functions and recently elucidated underlying molecular mechanism. In various neurodegenerative diseases, abnormal nuclear and cytoplasmic bodies are built on disease-causing RNAs or toxic RNAs with aberrantly expanded short tandem repeat-enriched sequences. We discuss the possible analogous functions of natural arcRNAs and toxic RNAs with short tandem repeat-enriched sequences. Finally, we describe the technical utility of short tandem repeat-enriched arcRNAs as a model for exploring the structures and functions of nuclear bodies, as well as the pathogenic mechanisms of neurodegenerative diseases.

Correlated cryogenic fluorescence microscopy and electron cryotomography shows that exogenous TRIM5α can form hexagonal lattices or autophagy aggregates in vivo

Members of the TRIM protein family have been shown to gather into structures in both the nucleus and cytoplasm. One TRIM protein family member, TRIM5α, has been shown to form cytoplasmic bodies involved in restricting retroviruses such as HIV-1. Here we applied cryogenic correlated light and electron microscopy (cryo-CLEM) to intact mammalian cells expressing YFP-rhTRIM5α and found hexagonal nets were present whose arm-lengths were similar to those of the hexagonal nets formed by purified TRIM5α in-vitro. We also observed YFP-rhTRIM5α within a diversity of structures with characteristics expected for organelles involved in different stages of macroautophagy, including disorganized protein aggregations (sequestosomes), sequestosomes flanked by flat double-membraned vesicles (sequestosome:phagophore complexes), sequestosomes within a double-membraned vesicle (autophagosomes), and sequestosomes within multi-vesicular autophagic vacuoles (autolysosomes or amphisomes). Vaults were also seen in these structures, consistent with their role in autophagy. Our data (i) support recent reports that TRIM5α can form both well-organized signaling complexes and non-signaling aggregates, (ii) offer the first images of the macroautophagy pathway in a near-native state, and (iii) reveal that vaults arrive early in macroautophagy.

TRIM34 acts with TRIM5 to restrict HIV and SIV capsids

The HIV-1 capsid protein makes up the core of the virion and plays a critical role in early steps of HIV replication. Due to its exposure in the cytoplasm after entry, HIV capsid is a target for host cell factors that act directly to block infection such as TRIM5 and MxB. Several host proteins also play a role in facilitating infection, including in the protection of HIV-1 capsid from recognition by host cell restriction factors. Through an unbiased screening approach, called HIV-CRISPR, we show that the Cyclophilin A-binding deficient P90A HIV-1 capsid mutant becomes highly-sensitized to TRIM5alpha restriction in IFN-treated cells. Further, the CPSF6-binding deficient, N74D HIV-1 capsid mutant is sensitive to restriction mediated by human TRIM34, a close paralog of the well-characterized HIV restriction factor TRIM5. This restriction occurs at the step of reverse transcription, is independent of interferon stimulation and limits HIV-1 infection in key target cells of HIV infection including CD4+ T cells and monocyte-derived dendritic cells. TRIM34 restriction requires TRIM5alpha as knockout or knockdown of TRIM5alpha results in a loss of antiviral activity. TRIM34 can also restrict some SIV capsids. Through immunofluorescence studies, we show that TRIM34 and TRIM5alpha colocalize to cytoplasmic bodies and are more frequently observed to be associated with infecting N74D capsids than with WT capsids. Our results identify TRIM34 as an HIV-1 CA-targeting restriction factor and highlight the potential role for heteromultimeric TRIM interactions in contributing restriction of HIV-1 infection in human cells.

Core spliceosomal Sm proteins as constituents of cytoplasmic mRNPs in plants

ABSTRACTIn light of recent studies, many of the cytoplasmic posttranscriptional mRNA processing steps take place in highly specialized microdomains referred to as cytoplasmic bodies. These evolutionarily conserved microdomains are sites of regulation for both mRNA translation and degradation. It has been shown that in the larch microsporocyte cytoplasm, there is a significant pool of Sm proteins not related to snRNP complexes. These Sm proteins accumulate within distinct cytoplasmic bodies (S-bodies) that also contain mRNA. Sm proteins constitute an evolutionarily ancient family of small RNA-binding proteins. In eukaryotic cells, these molecules are involved in pre-mRNA splicing. The latest research indicates that in addition to this well-known function, Sm proteins could also have an impact on mRNA at subsequent stages of its life cycle. The aim of this work was to verify the hypothesis that canonical Sm proteins are part of the cytoplasmic mRNP complex and thus function in the posttranscriptional regulation of gene expression in plants.

Clueless forms dynamic, insulin-responsive bliss particles sensitive to stress

Mitochondria perform a myriad of biochemical functions in the cell that integrate ATP production and metabolism. While mitochondria contain their own genome, mtDNA, it only encodes thirteen proteins required for oxidative phosphorylation, thus well over one thousand proteins required for all mitochondrial functions are encoded in the nucleus. One such protein is Drosophila Clueless (Clu), whose vertebrate homolog is Clustered mitochondria homolog (Cluh). Clu/Cluh is a ribonucleoprotein that regulates mRNAs destined for import into mitochondria and is an essential protein that regulates cellular metabolism. Clu forms large particles in the cytoplasm, although how these particles relate to nutrition and metabolic stress is unknown. Using live-imaging, we show Clu particles are highly dynamic. Clu particles appear to be unique as they do not colocalize with many known cytoplasmic bodies. In addition, Clu particle formation is highly dependent on diet as ovaries from starved females no longer contain Clu particles although Clu protein levels remain the same and insulin is necessary and sufficient for Clu particle formation. Oxidative stress also disperses particle. Since Clu particles are only present under optimal conditions we are naming them bliss particles. These observations identify Clu particles as unique, stress-sensitive cytoplasmic ribonucleoprotein particles whose absence corresponds with altered mitochondrial function and localization.

Studying Variation in Cell Apoptosis Caused by Corticosteroids in Thymus of the Rat

Background and purpose: Apoptosis or planned death of cells is the main mechanism in completion and hemostasis of mature tissues to terminate useless, contaminated, and mutated/damaged cells through internal suicide pathways. One of the agents that creates apoptosis signals is glucocorticoids. As the representative of corticosteroid drugs, dexamethasone can induce apoptosis via internal endonuclease. Therefore, dexamethasone was used as a synthetic glucocorticoid in rats. The effects of dexamethasone on thymocyte of rats, morphological features of apoptotic cells, and the relationship between drug dosage and severity of apoptosis were examined. Methodology: A treatment group with four subgroups (each with five rats) namely T-a, T-b- T-c, and T-a was formed and received IP dexamethasone of 0.5, 1.5, 2.5, 3.5mg per one-kilogram body weight respectively. In addition, a control group with similar subgroups was formed. Thymus gland of the subjects of both groups was removed six hours after injection. The samples were examined using electron and optical microscopes.Findings: Examinations by optical microscope revealed round or elliptic apoptotic cytoplasmic bodies with or without nucleus basophil materials along with crescentic bodies of chromatin on the apoptotic cells. In addition, electronic microscope images reveled marginal augmentation of nucleus chromatin in osmiophilic forms detached from nucleus fibrillary centers along with a disordered cellular region, endoplasmic reticulum complication, and nucleus fragmentation. There was a direct and significant relationship between the dosage of the drug and the severity of apoptosis.Conclusion and Recommendations: The results showed that standard and higher dosages of corticosteroid stimulated DNA fragmentation in thymocytes and apoptosis of thymocytes induced by corticosteroid is a calcium-depended process.