Theophany Eystathioy

Disruption of GW bodies impairs mammalian RNA interference.

The GW182 RNA-binding protein was initially shown to associate with a specific subset of mRNAs and to reside within discrete cytoplasmic foci named GW bodies (GWBs). GWBs are enriched in proteins that are involved in mRNA degradation. Recent reports have shown that exogenously introduced human Argonaute-2 (Ago2) is also enriched in GWBs, indicating that RNA interference function may be somehow linked to these structures. In this report, we demonstrate that endogenous Ago2 and transfected small interfering RNAs (siRNAs) are also present within these same cytoplasmic bodies and that the GW182 protein interacts with Ago2. Disruption of these cytoplasmic foci in HeLa cells interferes with the silencing capability of a siRNA that is specific to lamin-A/C. Our data support a model in which GW182 and/or the microenvironment of the cytoplasmic GWBs contribute to the RNA-induced silencing complex and to RNA silencing.

GW182 is critical for the stability of GW bodies expressed during the cell cycle and cell proliferation

A novel cytoplasmic compartment referred to as GW bodies was initially identified using human autoantibodies to a 182 kDa protein named GW182. GW bodies are small, generally spherical, cytoplasmic domains that vary in number and size in several mammalian cell types examined to date. Based on our earlier studies, GW bodies were proposed to be cytoplasmic sites for mRNA storage and/or degradation. In the present study, immunogold electron microscopy identified electron dense structures of 100-300 nm diameter devoid of a lipid bilayer membrane. These structures appeared to comprise clusters of electron dense strands of 8-10 nm in diameter. By costaining with CENP-F and PCNA, and employing a double-thymidine block to synchronize HeLa cells, GW bodies were observed to be small in early S phase and larger during late S and G2 phases of the cell cycle. The majority of GW bodies disassembled prior to mitosis and small GW bodies reassembled in early G1. The analysis of GW bodies in two experimental models of cell proliferation using reversal of 3T3/serum-starvation and concanavalin A stimulation of mouse splenocytes and T cells, revealed that proliferating cells contained larger, brighter, and more numerous GW bodies as well as up to a fivefold more total GW182 protein than quiescent cells. In vitro gene knockdown of GW182 led to the disappearance of GW bodies demonstrating that GW182 is a critical component of GW bodies. The incremental expression of the GW182 protein in cells induced to proliferate and the cyclic formation and breakdown of GW bodies during mitosis are intriguing in view of the notion that GW bodies are specialized centers involved in maintaining stability and/or controlling degradation of mRNA.

The role of GW/P-bodies in RNA processing and silencing

GW bodies, also known as mammalian P-bodies, are cytoplasmic foci involved in the post-transcriptional regulation of eukaryotic gene expression. Recently, GW bodies have been linked to RNA interference and demonstrated to be important for short-interfering-RNA- and microRNA-mediated mRNA decay and translational repression. Evidence indicates that both passenger and guide strands of short-interfering RNA duplexes can localize to GW bodies, thereby indicating that RNA-induced silencing complexes may be activated within these cytoplasmic centers. Formation of GW bodies appears to depend on both specific protein factors and RNA, in particular, microRNA. Work over the past few years has significantly increased our understanding of the biology of GW bodies, revealing that they are specialized cell components that spatially regulate mRNA turnover in various biological processes. The formation of GW bodies appears to depend on both specific protein factors and RNA, in particular, microRNA. Here, we propose a working model for GW body assembly in terms of its relationship to RNA interference. In this process, one or more heteromeric protein complexes accumulate in successive steps into larger ribonucleoprotein structures.

The orphan nuclear receptors BmE75A and BmE75C of the silkmoth Bombyx mori: hornmonal control and ovarian expression.

The steroid hormone 20-hydroxyecdysone (20E) plays a key role in the stimulation of ovarian follicle development in the silkmoth, Bombyx mori. To understand better the mechanism by which 20E regulates silkmoth oogenesis, Bombyx homologs of the ecdysone-inducible orphan nuclear receptor E75 (BmE75) were cloned and their expression was analyzed in developing ovaries and staged follicles during metamorphosis. Of the two BmE75 isoforms isolated, only the A-isoform (BmE75A) has been identified previously in lepidopteran insects. BmE75C, on the other hand, shows significant sequence homology in its N-terminus to the Drosophila E75C isoform. Northern blot analysis shows unique expression patterns for each isoform mRNA during ovarian development. While the A-isoform seems to be mainly implicated in the earlier stages of the ecdysone response during previtellogenesis and vitellogenesis, expression of the C-isoform becomes strongly induced in an ecdysteroid-independent fashion at the transition from vitellogenesis to choriogenesis. Our data indicate a complex regulation of the expression of the BmE75 gene during oogenesis and postulate a new role for the BmE75C receptor at the end of vitellogenesis and the beginning of choriogenesis.

GW bodies, microRNAs and the cell cycle.

GW bodies (GWBs) are cytoplasmic foci initially identified through the use of an autoimmuneserum targeting the marker protein, GW182. GWBs were first considered as both storagecenters for a specific subset of mRNAs and degradation sites for mRNAs. Interestingly, theyare known to vary in size and number throughout the cell cycle and are largest in size and mostabundant in number during the late S and G2 phases. Recent studies have linked RNAinterference to GWBs, in that disruption or disassembly of GWBs was demonstrated to impairsiRNA and miRNA silencing activity. As miRNAs are implicated in the regulation of cellcycle progression and cell proliferation, it is very likely that GWBs, the critical intracellularstructures for miRNA function, may very well be also linked to this cellular process.

The orphan nuclear receptor BmHR3A of Bombyx mori: hormonal control, ovarian expression and functional properties.

Ovarian development in the domesticated silkmoth, Bombyx mori, is induced by the molting hormone 20-hydroxy-ecdysone (20E) shortly after larval-pupal ecdysis. Studies of the ecdysone response in Drosophila and other insects have shown that 20E exerts its effects initially by the induction of a small number of early genes, including the orphan nuclear receptors HR3, that transduce and amplify the hormone signal. Here we show that the silkmoth orphan receptor BmHR3A acts in the 20E-induced regulatory cascade in the ovary during pupal and pharate adult development in a manner different than that observed in the classical ecdysone regulatory hierarchy in Drosophila salivary glands at the end of the third instar. While other isoforms of BmHR3 are induced as early gene products in the ecdysone response, BmHR3A is induced 2 days after 20E administration in the silkmoth ovary and, thus, behaves as late product. The period of accumulation of BmHR3A in ovarian follicular cells occurs during vitellogenesis and coincides with the period of transcriptional expression of the ESP (egg-specific protein) gene, whose product constitutes a major component of the egg yolk, while it is reciprocal to the period of expression of BmGATAbeta, a gene encoding a regulator of late chorion gene expression. Bandshift experiments demonstrate that BmHR3A binds specifically to RORE (Retinoic acid-related Orphan receptor Response Element)-like sequences in the promoters of both genes, thus suggesting a direct role for BmHR3A in regulating the expression of BmGATAbeta and ESP genes during vitellogenesis. Finally, we show that BmHR3A functions as a constitutive transcriptional activator in a B. mori derived cell line. We propose that BmHR3A may function as a regulator of vitellogenesis in the silkmoth ovary.

Markers of mRNA stabilization and degradation, and RNAi within astrocytoma GW bodies.

GW bodies (GWBs) are unique cytoplasmic structures that contain the mRNA binding protein GW182 and other proteins involved in mRNA processing pathways. The rationale for this study arose from clinical studies indicating that 33% of patients with GWB autoantibodies have a motor/sensory neuropathy and/or ataxia. The novelty of this study is the identification of GWBs in astrocytes and astrocytoma cells within cell bodies and cytoplasmic projections. Astrocytoma GWBs exhibit complex heterogeneity with combinations of LSm4 and XRN1 as well as Ago2 and Dicer, key proteins involved in mRNA degradation and RNA interference, respectively. GWB subsets contained the mRNA transport and stabilization proteins SYNCRIP, hnRNPA1, and FMRP, not previously described as part of the GWB complex. Immunoprecipitation of astrocytoma GWBs suggested that Dicer, hDcp, LSm4, XRN1, SYNCRIP, and FMRP form a multiprotein complex. GWBs are likely involved in a number of regulatory mRNA pathways in astrocytes and astrocytoma cells.

A panel of monoclonal antibodies to cytoplasmic GW bodies and the mRNA binding protein GW182.

GW182 is a mRNA binding protein characterized by 60 repeats of glycine (G):tryptophan (W) motifs and is localized in cytoplasmic structures referred to as GW bodies (GWBs). Current evidence suggests that this unique protein plays a role in mRNA processing. To enable a more detailed study of GW182 and GWBs in cells and tissues, including their role in mRNA processing, we developed four monoclonal antibodies (MAbs) that bind the human recombinant GW182 protein. These MAbs can be used for Western blot analysis and indirect immunofluorescence (IIF) on cultured cells and tissues. Of special interest, one of the MAbs, 2D6, can be used to identify GW182 and GWBs in formalin-fixed and paraffin-embedded tissues after using an antigen retrieval method (ARM). All the MAbs described in this study immunoprecipitate the GW182 protein. Epitope mapping using overlapping 15-mer peptides representing the full-length GW182 showed that the major antibody-binding domains of these MAbs are distinct. These MAbs are valuable tools for cell biologists and pathologists to study the location and function of the novel GW182 protein in tissue culture cells, as well as cryopreserved or archived tissues.

GW Bodies: Cytoplasmic Compartments in Normal Human Skin

TO THE EDITOR In 2002, unique cytoplasmic compartments were identified and termed GW bodies (GWBs), also known as mammalian processing bodies (P bodies) (Eystathioy et al., 2002; van Dijk et al., 2002; Sheth and Parker, 2003; Eulalio et al., 2007; Jakymiw et al., 2007; Parker and Sheth, 2007). GWBs were named after the GW182 protein, an mRNA-binding protein that resides within these macromolecular foci (Eystathioy et al., 2002). Characterization of this protein revealed numerous GW (glycine–tryptophan) repeats and a canonical RNA recognition motif (Eystathioy et al., 2002). The GW182 protein associates with a specific subset of mRNA, serves as a scaffold or matrix protein, and helps to maintain the integrity of GWBs as knock down of the GW182 protein results in a loss of GWBs (Jakymiw et al., 2005, 2007). GWBs contain a defined subset of mRNA, microRNA, and various proteins involved in mRNA degradation and RNA interference (RNAi) (Eystathioy et al., 2003; Andrei et al., 2005; Jakymiw et al., 2005; Liu et al., 2005a, b; Rehwinkel et al., 2005; Behm-Ansmant et al., 2006; Pauley et al., 2006; Valencia-Sanchez et al., 2006). Intact GWBs are also found to be critical to the RNAi pathway, and microRNA is critical in the formation of GWBs (Jakymiw et al., 2005; Pauley et al., 2006). Lastly, GWBs have been shown to interact with stress granules during cellular stress responses (Kedersha et al., 2005). Current research has focused on improving our understanding of the structure and function of GWBs in a variety of cells and tissues during embryonic development, physiological responses, neoplastic transformation, and aging. As these structures function to localize specific subsets of mRNA and microRNAs, degrade mRNAs, interact with stress granules during the stress response, and are differentially regulated during the cell cycle and cell proliferation, GWBs may serve as important regulatory sites for normal, inflammatory, and malignant skin disease. To investigate the presence and distribution patterns of GWBs in normal human skin, 8-mm-thick paraffin-embedded normal skin sections were treated with an antigen retrieval method and stained with mouse mAbs to the recombinant GW182 protein as previously described (Jakymiw et al., 2005). In the epidermis and cutaneous appendages, GWBs were most prominent in the stratum basale and the outer root sheath of the hair follicle (Figure 1a–d). The GWBs visualized in these structures were generally larger and more numerous than in other cutaneous structures. In the epidermis, they were predominantly located in a single row along the basal layer, although transverse sections revealed several layers of keratinocytes containing GWBs in the deep rete ridges. Colocalization studies with a rabbit polyclonal antibody to keratin 15 (ProteinTech Group Inc, Chicago, IL) confirmed the follicular localization of GWBs to bulge progenitor cells (Lyle et al., 1998; Morris et al., 2004; Ohyama et al., 2006) (Figure S1). GW bodies were also visualized in the basaloid cells of the sebaceous glands and the eccrine glands (Figure S2). The germinative matrix of the hair bulb

Autoantibodies to novel cytoplasmic structures (GW bodies) involved in mRNA processing

This particular cytoplasmic discrete speckled pattern (1,2) is quite unique as the structures observed are a new entity named GW bodies (Figure 1), abbreviated GWBs (also known as mammalian P bodies or processing bodies). Functionally, GWBs are involved in the process of RNA interference (RNAi), mRNA degradation and/or mRNA storage. A subset of mRNAs are targeted to these cytoplasmic structures, therefore GWBs play a central role in the cell by influencing the fate of mRNAs. GWBs are ubiquitous and present in many tissues and cell lines as well as normal cells but appear to be more highly expressed in cancer cells such as HEp-2 (laryngeal carcinoma) and HeLa (cervical carcinoma) that are now routinely used to screen human sera for the presence of autoantibodies. To date, five target autoantigens in GWBs have been published. They include GW182, a unique protein characterized by a numerous glycine (G) and tryptophan (W) repeats(3), Ge-1/hedls (4,5), RAP-55(6), diacyl-phosphatidyl ethanolamine(2), and Su/hAgo2(7). The clinical diagnosis of patients with autoantibodies to GWBs can be divided into four main groups. In a study from the University of Calgary, the most common diagnosis is Sjogren’s syndrome, systemic lupus erythematosus (SLE), and the third group has neurological disease(3). In another study from Brazil(2) there was no clear cut clinical associations, although 3 patients with SLE were noted, but the majority comprise the fourth category of other conditions(2). The difference between the clinical profiles in these two centers may be explained on the basis of different clinical referral patterns. For example, in Calgary, serum samples of patients with suspected autoimmune neurological disease (ataxia, motor and sensory neuropathy) are routinely evaluated. Examination of 5000 patient serum samples received during 2000-2001, by ANA at the Mitogen Advanced Diagnostics Laboratory at the University of Calgary, demonstrated that the prevalence Figura 1 – Hep2000 cells stained with patient serum containing autoantibodies to GWBs (red). The nuclei are stained with DAPI (blue). Arrows are pointing to different sized GWBs while the arrowhead marks a cell undergoing mitosis.