Cytoplasmic P-bodies Research Articles

The role of ER exit sites in maintaining P-body organization and transmitting ER stress response during Drosophila melanogaster oogenesis.

Processing bodies (P-bodies) are cytoplasmic membrane-less organelles which host multiple mRNA processing events. While the fundamental principles of P-body organization are beginning to be elucidated in vitro, a nuanced understanding of how their assembly is regulated in vivo remains elusive. Here, we investigate the potential link between ER exit sites and P-bodies in Drosophila melanogaster egg chambers. Employing a combination of live and super-resolution imaging, we found that P-bodies associated with ER exit sites are larger and less mobile than cytoplasmic P-bodies, indicating that they constitute a distinct class of P-bodies which are more mature than their cytoplasmic counterparts. Moreover, we demonstrate that altering the composition of ER exit sites has differential effects on core P-body proteins (Me31B, Cup, and Trailer Hitch) suggesting a potential role for ER exit sites in P-body organization. We further show that in the absence of ER exit sites, P-body integrity is compromised and the stability and translational repression efficiency of the maternal mRNA, oskar, are reduced. Finally, we show that ER stress is communicated to P-bodies via ER exit sites, highlighting the pivotal role of ER exit sites as a bridge between membrane-bound and membrane-less organelles in ER stress response. Together, our data unveils the significance of ER exit sites not only in governing P-body organization, but also in facilitating inter-organellar communication during stress, potentially bearing implications for a variety of disease pathologies.

Variable penetrance of Nab3 granule accumulation quantified by a new tool for high-throughput single-cell granule analysis.

Reorganization of cellular proteins into subcellular compartments, such as the concentration of RNA-binding proteins into cytoplasmic stress granules and P-bodies, is a well-recognized, widely studied physiological process currently under intense investigation. One example of this is the induction of the yeast Nab3 transcription termination factor to rearrange from its pan-nucleoplasmic distribution to a granule at the nuclear periphery in response to nutrient limitation. Recent work in many cell types has shown that protein condensation in the nucleus is functionally important for transcription initiation, RNA processing, and termination. However, little is known about how subnuclear compartments form. Here, we have quantitatively analyzed this dynamic process in living yeast using a high-throughput computational tool and fluorescence microscopy. This analysis revealed that Nab3 granule accumulation varies in penetrance across yeast strains. A concentrated single granule is formed from at least a quarter of the nuclear Nab3 drawn from the rest of the nucleus. Levels of granule accumulation were inversely correlated with a growth defect in the absence of glucose. Importantly, the basis for some of the variation in penetrance was attributable to a defect in mitochondrial function. This publicly available computational tool provides a rigorous, reproducible, and unbiased examination of Nab3 granule accumulation that should be widely applicable to a variety of fluorescent images. Thousands of live cells can be readily examined enabling rigorous statistical verification of significance. With it, we describe a new feature of inducible subnuclear compartment formation for RNA-binding transcription factors and an important determinant of granule biogenesis.

Pat1 RNA-binding proteins: Multitasking shuttling proteins.

Post-transcriptional regulation of gene expression is largely achieved at the level of splicing in the nucleus, and translation and mRNA decay in the cytosol. While the regulation may be global, through the direct inhibition of central factors, such as the spliceosome, translation initiation factors and mRNA decay enzymes, in many instances transcripts bearing specific sequences or particular features are regulated by RNA-binding factors which mobilize or impede recruitment of these machineries. This review focuses on the Pat1 family of RNA-binding proteins, conserved from yeast to man, that enhance the removal of the 5' cap by the decapping enzyme Dcp1/2, leading to mRNA decay and also have roles in translational repression. Like Dcp1/2, other decapping coactivators, including DDX6 and Edc3, and translational repressor proteins, Pat1 proteins are enriched in cytoplasmic P-bodies, which have a principal role in mRNA storage. They also concentrate in nuclear Cajal-bodies and splicing speckles and in man, impact splice site choice in some pre-mRNAs. Pivotal to these functions is the association of Pat1 proteins with distinct heptameric Lsm complexes: the cytosolic Pat1/Lsm1-7 complex mediates mRNA decay and the nuclear Pat1/Lsm2-8 complex alternative splicing. This dual role of human Pat1b illustrates the power of paralogous complexes to impact distinct processes in separate compartments. The review highlights our recent findings that Pat1b mediates the decay of AU-rich mRNAs, which are particularly enriched in P-bodies, unlike the decapping activator DDX6, which acts on GC-rich mRNAs, that tend to be excluded from P-bodies, and discuss the implications for mRNA decay pathways. This article is categorized under: RNA Turnover and Surveillance > Regulation of RNA Stability RNRNA Processing > Splicing Regulation/Alternative Splicing Translation > Translation Regulation.

Characterization of the Es-DDX52 involved in the spermatogonial mitosis and spermatid differentiation in Chinese mitten crab (Eriocheir sinensis)

Spermatogenesis involves a series of process including exiting from the mitotic cell cycle, entry into meiosis, completion of complex differentiation programs, and producing spermatozoa. Expression of various genes in an ordered manner, and interactions between various genes and their protein products, primarily controlled at the post-transcriptional level with DEAD-box RNA helicases playing a crucial role in germ cell development, are required for production of fertile sperm. Many members of this family have been deeply studied in spermatogenesis, such as DDX3X, DDX25 and DDX4, but few data are available on DDX52. In this study, we analyzed the expression patterns of Es-DDX52, Es-DDX6, Es-Vasa and Es-XRN1 both at mRNA and protein levels in different tissues and during gonadal development. It showed that Es-vasa, Es-DDX6 and Es-Xrn1, components of cytoplasmic foci P-bodies, have the similar transcriptional expression pattern, while Es-DDX52 has the reverse tendency. Furthermore, Es-DDX6 and Es-XRN1 proteins have the same localization in testicular tissues. Es-DDX52 mainly distributed in the cytoplasm of spermatogonia, only localized in the nucleus of early and middle spermatid and shifted to pre-acrosome vesicle (later developed into apical cap and acrosome tube) at both mRNA and protein levels. These results indicated that Es-DDX52 may participate in regulation of P-bodies and microtubules by Es-XRN1, and involved in the mitosis of spermatonia and spermatid differentiation.

CSN1 Somatic Mutations in Penile Squamous Cell Carcinoma.

Other than an association with HPV infection, little is known about the genetic alterations determining the development of penile cancer. Although penile cancer is rare in the developed world, it presents a significant burden in developing countries. Here, we report the findings of whole-exome sequencing (WES) to determine the somatic mutational landscape of penile cancer. WES was performed on penile cancer and matched germline DNA from 27 patients undergoing surgical resection. Targeted resequencing of candidate genes was performed in an independent 70 patient cohort. Mutation data were also integrated with DNA methylation and copy-number information from the same patients. We identified an HPV-associated APOBEC mutation signature and an NpCpG signature in HPV-negative disease. We also identified recurrent mutations in the novel penile cancer tumor suppressor genes CSN1(GPS1) and FAT1 Expression of CSN1 mutants in cells resulted in colocalization with AGO2 in cytoplasmic P-bodies, ultimately leading to the loss of miRNA-mediated gene silencing, which may contribute to disease etiology. Our findings represent the first comprehensive analysis of somatic alterations in penile cancer, highlighting the complex landscape of alterations in this malignancy. Cancer Res; 76(16); 4720-7. ©2016 AACR.

LINE-1 Retroelements Get ZAPped!

LINE-1 Retroelements Get ZAPped!

Prolonged Pseudohypoxia Targets Ambra1 mRNA to P-Bodies for Translational Repression.

Hypoxia has been associated with several pathological conditions ranging from stroke to cancer. This condition results in the activation of autophagy, a cyto-protective response involving the formation of double-membraned structures, the autophagosomes, in the cytoplasm. In this study, we investigated the cellular mechanisms regulating the autophagy gene Ambra1, after exposure to a hypoxia mimetic, cobalt chloride (CoCl2). We observed that, upon CoCl2 administration, activation of the apoptotic machinery was concomitant with down-regulation of the pro-autophagic factor Ambra1, without affecting transcription. Additionally, co-treating the cells with the caspase inhibitor z-VAD-FMK did not restore Ambra1 protein levels, this implying the involvement of other regulatory mechanisms. Partial re-localization of Ambra1 mRNA to non-translating fractions and cytoplasmic P-bodies was further detected. Thus, in this pseudohypoxic context, Ambra1 mRNA translocation to P-bodies and translational suppression correlated with increased cell death.

Proteomic analysis identifies endoribouclease EhL-PSP and EhRRP41 exosome protein as novel interactors of EhCAF1 deadenylase.

This study provides evidences for the functional deadenylase activity of EhCAF1 and shows a link between different mRNA degradation proteins in E. histolytica. By proteomic tools and pull down assays, we evidenced that EhCAF1 interacts with the putative endoribonuclease EhL-PSP, which in turn interacts with exosome EhRRP41 protein. Our data suggest for the first time the presence of two complexes, one containing the endoribonuclease EhL-PSP and the deadenylase EhCAF1 in P-bodies; and another containing the endoribonuclease EhL-PSP and the exosome EhRRP41 exoribonuclease. Overall, these results provide novel data that may help to understand mRNA decay mechanisms in this parasite.

Abstract 1: Role of MicroRNAs in Postranscriptional Regulation of Apolipoprotein B-100 mRNA

Hepatic apolipoprotein B-100 (apoB) synthesis and secretion appears to be regulated largely at the posttranscriptional and posttranslational levels. MicroRNAs (miRNAs) are among posttranscriptional regulators of gene expression that bind to complementary sequences on target messenger RNA (mRNA) transcripts, usually resulting in translational repression or degradation. It is unknown whether specific miRNAs are involved in posttranscriptional regulation of apoB mRNA. We performed bioinformatic analysis, showing that two specific miRNAs with satisfactory E-values level (with levels indicating greater similarity between the input and its match) namely, miR-544 (E-value = 0.91) and miR-1202 (E-value=0.86) have potential to interact with 3’ and 5’ UTR of apoB, respectively. We hypothesized that the interaction of these specific miRNAs (miR-544 and miR-1202) with the 3’ and 5’UTR of apoB mRNA leads to apoB mRNA translational repression and/or activation. Using a human hepatoma cell line model, HepG2, the effects of overexpressed miRNAs and inhibition of endogenous miRNAs on the expression of apoB mRNA and apoB protein synthesis were investigated. We further examined the effect of these miRNAs on apoB mRNA traffic into cytoplasmic P-bodies. Transfection of HepG2 cells with miR-544 led to a significant reduction in apoB mRNA expression and protein synthesis and induced an increase in the co-localization of apoB mRNA into P-bodies. The opposite effect was observed when anti-miR-544 was employed to inhibit the endogenous miR-544. Results from luciferase reporter assays indicated that the effects of miR-544 may be mediated via interaction with the 3’UTR of apoB mRNA. In contrast to miR-544, miR-1202 overexpression induced an increase in apoB mRNA expression and protein synthesis. Similarly, the opposite effect was observed when using anti-miR-1202. Data from luciferase reporter assays showed an increased expression of the reporter gene in constructs carrying 5’UTR of apoB mRNA suggesting that miR-1202 may function via the 5’UTR. In summary, these data demonstrate that specific miRNAs are involved in the regulation of expression and translational control of apoB mRNA in hepatocytes. However, these miRNAs do not appear to mediate insulin regulation.

MRNA Decapping Enzyme 1a (Dcp1a)-induced Translational Arrest through Protein Kinase R (PKR) Activation Requires the N-terminal Enabled Vasodilator-stimulated Protein Homology 1 (EVH1) Domain

We have shown previously that poliovirus infection disrupts cytoplasmic P-bodies in infected mammalian cells. During the infectious cycle, poliovirus causes the directed cleavage of Dcp1a and Pan3, coincident with the dispersion of P-bodies. We now show that expression of Dcp1a prior to infection, surprisingly, restricts poliovirus infection. This inhibition of infection was independent of P-body formation because expression of GFP-Dcp1a mutants that cannot enter P-bodies restricted poliovirus infection similar to wild-type GFP-Dcp1a. Expression of wild-type or mutant GFP-Dcp1a induced phosphorylation of eIF2α through the eIF2α kinase protein kinase R (PKR). Activation of PKR required the amino-terminal EVH1 domain of Dcp1a. This PKR-induced translational inhibition appears to be specific to Dcp1a because the expression of other P-body components, Pan2, Pan3, Ccr4, or Caf1, did not result in the inhibition of poliovirus gene expression or induce eIF2α phosphorylation. The translation blockade induced by Dcp1a expression suggests novel signaling linking RNA degradation/decapping and regulation of translation.

DDX3 RNA helicase is required for HIV-1 Tat function

Host RNA helicase has been involved in human immunodeficiency virus type 1 (HIV-1) replication, since HIV-1 does not encode an RNA helicase. Indeed, DDX1 and DDX3 DEAD-box RNA helicases are known to be required for efficient HIV-1 Rev-dependent RNA export. However, it remains unclear whether DDX RNA helicases modulate the HIV-1 Tat function. In this study, we demonstrate, for the first time, that DDX3 is required for the HIV-1 Tat function. Notably, DDX3 colocalized and interacted with HIV-1 Tat in cytoplasmic foci. Indeed, DDX3 localized in the cytoplasmic foci P-bodies or stress granules under stress condition after the treatment with arsenite. Importantly, only DDX3 enhanced the Tat function, while various distinct DEAD-box RNA helicases including DDX1, DDX3, DDX5, DDX17, DDX21, and DDX56, stimulated the HIV-1 Rev-dependent RNA export function, indicating a specific role of DDX3 in Tat function. Indeed, the ATPase-dependent RNA helicase activity of DDX3 seemed to be required for the Tat function as well as the colocalization with Tat. Furthermore, the combination of DDX3 with other distinct DDX RNA helicases cooperated to stimulate the Rev but not Tat function. Thus, DDX3 seems to interact with the HIV-1 Tat and facilitate the Tat function.

The P-body component USP52/PAN2 is a novel regulator of HIF1A mRNA stability

HIF1A (hypoxia-inducible factor 1α) is the master regulator of the cellular response to hypoxia and is implicated in cancer progression. Whereas the regulation of HIF1A protein in response to oxygen is well characterized, less is known about the fate of HIF1A mRNA. In the present study, we have identified the pseudo-DUB (deubiquitinating enzyme)/deadenylase USP52 (ubiquitin-specific protease 52)/PAN2 [poly(A) nuclease 2] as an important regulator of the HIF1A-mediated hypoxic response. Depletion of USP52 reduced HIF1A mRNA and protein levels and resulted in reduced expression of HIF1A-regulated hypoxic targets due to a 3′-UTR (untranslated region)-dependent poly(A)-tail-length-independent destabilization in HIF1A mRNA. MS analysis revealed an association of USP52 with several P-body (processing body) components and we confirmed further that USP52 protein and HIF1A mRNA co-localized with cytoplasmic P-bodies. Importantly, P-body dispersal by knockdown of GW182 or LSM1 resulted in a reduction of HIF1A mRNA levels. These data uncover a novel role for P-bodies in regulating HIF1A mRNA stability, and demonstrate that USP52 is a key component of P-bodies required to prevent HIF1A mRNA degradation.

Acidic stress induces the formation of P-bodies, but not stress granules, with mild attenuation of bulk translation inSaccharomyces cerevisiae

The stress response of eukaryotic cells often causes an attenuation of bulk translation activity and the accumulation of non-translating mRNAs into cytoplasmic mRNP (messenger ribonucleoprotein) granules termed cytoplasmic P-bodies (processing bodies) and SGs (stress granules). We examined effects of acidic stress on the formation of mRNP granules compared with other forms of stress such as glucose deprivation and a high Ca²⁺ level in Saccharomyces cerevisiae. Treatment with lactic acid clearly caused the formation of P-bodies, but not SGs, and also caused an attenuation of translation initiation, albeit to a lesser extent than glucose depletion. P-body formation was also induced by hydrochloric acid and sulfuric acid. However, lactic acid in SD (synthetic dextrose) medium with a pH greater than 3.0, propionic acid and acetic acid did not induce P-body formation. The results of the present study suggest that the assembly of yeast P-bodies can be induced by external conditions with a low pH and the threshold was around pH 2.5. The P-body formation upon acidic stress required Scd6 (suppressor of clathrin deficiency 6), a component of P-bodies, indicating that P-bodies induced by acidic stress have rules of assembly different from those induced by glucose deprivation or high Ca²⁺ levels.

Sequestration of Highly Expressed mRNAs in Cytoplasmic Granules, P-Bodies, and Stress Granules Enhances Cell Viability

Transcriptome analyses indicate that a core 10%–15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress.

Direct evidence that RNA inhibits APOBEC3G ssDNA cytidine deaminase activity

APOBEC3G (A3G) is a deoxycytidine deaminase active on ssDNA substrates. In HIV infected cells A3G interacted with reverse transcription complexes where its activity as a deoxycytidine deaminase led to mutation of the viral genome. A3G not only bound ssDNA, but it also had an intrinsic ability to bind RNA. In many cell types that can support HIV replication, A3G ssDNA deaminase activity was suppressed and the enzyme resided in high molecular mass, ribonucleoprotein complexes associated with cytoplasmic P-bodies and stress granules. Using a defined in vitro system, we show that RNA alone was sufficient to suppress A3G deaminase activity and did so in an RNA concentration-dependent manner. RNAs of diverse sequences and as short as 25nt were effective inhibitors. Native PAGE analyses showed that RNA formed ribonucleoprotein complexes with A3G and in so doing prevented ssDNA substrates from binding to A3G. The data provided direct evidence that A3G binding to cellular RNAs constituted a substantial impediment to the enzyme’s ability to interact with ssDNA.

Microtubule disruption targets HIF-1α mRNA to cytoplasmic P-bodies for translational repression

The hypoxia inducible factor 1α (HIF-1α) is overexpressed in solid tumors, driving tumor angiogenesis and survival. However, the mechanisms regulating HIF-1α expression in solid tumors are not fully understood. In this study, we find that microtubule integrity and dynamics are intricately involved in orchestrating HIF-1α translation. HIF-1α messenger RNA (mRNA) traffics on dynamic microtubules when it is actively translated. Microtubule perturbation by taxol (TX) and other microtubule-targeting drugs stalls HIF-1α mRNA transport and releases it from polysomes, suppressing its translation. Immunoprecipitation of the P-body component Argonaute 2 (Ago2) after microtubule disruption shows significant enrichment of HIF-1α mRNAs and HIF-targeting microRNAs (miRNAs). Inhibition of HIF-repressing miRNAs or Ago2 knockdown abrogates TX's ability to suppress HIF-1α translation. Interestingly, microtubule repolymerization after nocodazole washout allows HIF-1α mRNA to reenter active translation, suggesting that microtubule dynamics exert tight yet reversible control over HIF-1α translation. Collectively, we provide evidence for a new mechanism of microtubule-dependent HIF-1α translation with important implications for cell biology.

A Role for Huntington Disease Protein in Dendritic RNA Granules

Regulated transport and local translation of mRNA in neurons are critical for modulating synaptic strength, maintaining proper neural circuitry, and establishing long term memory. Neuronal RNA granules are ribonucleoprotein particles that serve to transport mRNA along microtubules and control local protein synthesis in response to synaptic activity. Studies suggest that neuronal RNA granules share similar structures and functions with somatic P-bodies. We recently reported that the Huntington disease protein huntingtin (Htt) associates with Argonaute (Ago) and localizes to cytoplasmic P-bodies, which serve as sites of mRNA storage, degradation, and small RNA-mediated gene silencing. Here we report that wild-type Htt associates with Ago2 and components of neuronal granules and co-traffics with mRNA in dendrites. Htt was found to co-localize with RNA containing the 3'-untranslated region sequence of known dendritically targeted mRNAs. Knockdown of Htt in neurons caused altered localization of mRNA. When tethered to a reporter construct, Htt down-regulated reporter gene expression in a manner dependent on Ago2, suggesting that Htt may function to repress translation of mRNAs during transport in neuronal granules.

Parainfluenza virus 5 genomes are located in viral cytoplasmic bodies whilst the virus dismantles the interferon-induced antiviral state of cells

Although the replication cycle of parainfluenza virus type 5 (PIV5) is initially severely impaired in cells in an interferon (IFN)-induced antiviral state, the virus still targets STAT1 for degradation. As a consequence, the cells can no longer respond to IFN and after 24−48 h, they go out of the antiviral state and normal virus replication is established. Following infection of cells in an IFN-induced antiviral state, viral nucleocapsid proteins are initially localized within small cytoplasmic bodies, and appearance of these cytoplasmic bodies correlates with the loss of STAT1 from infected cells. In situ hybridization, using probes specific for the NP and L genes, demonstrated the presence of virus genomes within these cytoplasmic bodies. These viral cytoplasmic bodies do not co-localize with cellular markers for stress granules, cytoplasmic P-bodies or autophagosomes. Furthermore, they are not large insoluble aggregates of viral proteins and/or nucleocapsids, as they can simply and easily be dispersed by ‘cold-shocking’ live cells, a process that disrupts the cytoskeleton. Given that during in vivo infections, PIV5 will inevitably infect cells in an IFN-induced antiviral state, we suggest that these cytoplasmic bodies are areas in which PIV5 genomes reside whilst the virus dismantles the antiviral state of the cells. Consequently, viral cytoplasmic bodies may play an important part in the strategy that PIV5 uses to circumvent the IFN system.

Nhl-2 Modulates MicroRNA Activity in Caenorhabditis elegans

TRIM-NHL proteins represent a large class of metazoan proteins implicated in development and disease. We demonstrate that a C. elegans TRIM-NHL protein, NHL-2, functions as a cofactor for the microRNA-induced silencing complex (miRISC) and thereby enhances the posttranscriptional repression of several genetically verified microRNA targets, including hbl-1 and let-60/Ras (by the let-7 family of microRNAs) and cog-1 (by the lsy-6 microRNA). NHL-2 is localized to cytoplasmic P-bodies and physically associates with the P-body protein CGH-1 and the core miRISC components ALG-1/2 and AIN-1. nhl-2 and cgh-1 mutations compromise the repression of microRNA targets in vivo but do not affect microRNA biogenesis, indicating a role for an NHL-2:CGH-1 complex in the effector phase of miRISC activity. We propose that the NHL-2:CGH-1 complex functions in association with mature miRISC to modulate the efficacy of microRNA:target interactions in response to physiological and developmental signals, thereby ensuring the robustness of genetic regulatory pathways regulated by microRNAs.

Phosphorylation of Argonaute proteins: regulating gene regulators

Members of the Ago (Argonaute) protein family are the mediators of small RNA-guided gene-silencing pathways including RNAi (RNA interference), translational regulation by miRNAs (microRNAs) and transcriptional silencing. Recent findings by Zeng et al. in this issue of the Biochemical Journal demonstrate that Ago proteins are post-translationally modified by phosphorylation of Ser(387). Mutating Ser(387) to alanine leads to reduced localization of human Ago2 to cytoplasmic P-bodies (processing bodies), cellular sites where RNA turnover and, at least in part, miRNA-guided gene regulation occurs. Zeng et al. further show that a member of the MAPK (mitogen-activated protein kinase) signalling pathway phosphorylates Ago2 at Ser(387), suggesting that Ago2-mediated gene silencing might be linked to distinct signalling pathways.