We have identified a novel protein that we have named GW182 because of its multiple glycine-tryptophan repeats found throughout the protein and the predicted and observed molecular mass of 182 kDa. By IIF, antibodies to native and recombinant GW182 appear to identify a cytoplasmic domain that we have tentatively named GWBs. The contention that GW182 resides in a distinct cytoplasmic domain is supported by the observation that antibodies to GW182 did not colocalize with markers of the Golgi complex, endosomes, lysosomes, or peroxisomes. Although there may appear to be a few overlaps with of some of these cytoplasmic markers, the overall IIF patterns observed with these markers and the IIF staining by the prototype human serum or rabbit anti-GW182p are clearly very different. Another possibility is that the GWBs might represent aggresomes or sites of protein degradation. However, our experiments using proteosome inhibitors or a target of the SCF ubiquitin ligase–mediated pathway for protein degradation suggests that GWBs are not aggresomes (Garcia-Mata et al., 1999
; Johnston et al., 2000
). Last, the observation that GWBs are not colocalized to sites marked by anticlathrin suggests that these bodies are probably not involved in clathrin-dependent vesicle transport (Hirst and Robinson, 1998
). These conclusions are supported by IEM data showing that anti-GW182 identifies a granular, electron-dense body in the cytoplasm of HeLa cells that is the same size as that identified by IIF and colocalization studies.
The data showing that the GW182 protein binds to several different mRNA species is quite intriguing. The lack of colocalization of GWBs with various known cytoplasmic organelles may not be surprising considering the interaction of GW182 with mRNA. Perhaps the GW182 protein is involved in stabilizing and/or regulating translation and/or storing mRNAs. Several mRNAs as members of mRNP complexes have been identified using antibodies to RNA proteins such as ELAV/Hu, elf-4E, and poly(A) binding proteins (Tenenbaum et al., 2000
; Keene, 2001
). ELAV/Hu proteins are involved in stabilizing and/or regulating translation of early response gene transcripts expressed primarily in neurons (Keene, 1999
; Brennan and Steitz, 2001
). Analysis of the transcripts revealed some limited sequence similarity, suggesting that a common theme exists among these transcripts that allows recognition by ELAV/Hu proteins. Interestingly, ELAV/Hu proteins along with a fraction of polyadenylated mRNA have been observed in discrete clusters within the cytoplasm of medulloblastoma cells (Antic and Keene, 1998
). It is possible that transcripts may be clustered in vivo with similar fates and/or functions. For example, upon retinoic acid treatment of P19 cells to induce neuronal differentiation, the population of mRNAs identified in the ELAV/Hu protein complex changed; additional mRNAs with AU-rich elements known to be upregulated in neurons were found (Tenenbaum et al., 2000
At the present time, we have not identified common functional or structural features among the mRNAs that are immunoprecipitated by the anti-GW182 antibodies. Some of the proteins are known to be key components of the cell cycle, whereas others have no immediately apparent relationship with one other. New approaches to elucidate unique structural features of mRNA are being developed and may shed light on the role of GW182 and related proteins. We are pursuing in situ hybridization using the mRNAs identified in this study and appropriate controls to determine if these mRNAs are located in GWBs.
In general the various functions of the cell are highly coordinated and regulated, and among these functions, gene transcription and translation are also highly regulated. The movement of mRNA transcripts into the cytoplasm and subsequent coordinated translation after appropriate endogenous or exogenous signals would necessarily involve highly sophisticated levels of control (Keene, 1999
; Tenenbaum et al., 2000
; Keene, 2001
). For example, some mRNAs may be required for rapid protein production and may be stored or protected from degradation. Because the cytoplasmic domains observed by IIF using the index human sera and rabbit anti-GW182 antibody failed to colocalize with markers of several cytoplasmic organelles and because of our observation that specific mRNAs are bound by GW182, it seems reasonable to conclude that GW182 is involved in mRNA expression and that this occurs in a defined cytoplasmic domain that we identify as GWBs. It might also be that GW182 is involved in degradation of mRNA but GWBs do not colocalize with the SCF ubiquitin ligase-mediated pathway for protein degradation.
Along these lines of discussion, the GWBs described in this study may be related to mRNA-associated particles described in other systems, particularly neuronal cells (Triedge et al., 1991
; Miyashiro et al., 1994
; Knowles et al., 1996
; Martone et al., 1996
; Gazzaley et al., 1997
; Racca et al., 1997
; Bassell et al., 1998
; Steward et al., 1998
). In oligodendrocytes, injected fluorescent-labeled myelin basic protein mRNA localized to granules that had a radius of 0.6–0.8 μm, contained elongation factors, rRNA and other mRNAs. It was concluded that these granules may represent supramolecular complexes of a translational unit (Barbarese et al., 1995
; Ainger et al., 1997
). These observations are consistent with the size of HeLa and HEp-2 cell granules observed in our study and suggest that GW182 marks a subset of these cytoplasmic bodies that contain a distinct subset of mRNAs. RNA granules containing mRNAs have also been described in fibroblasts (Ross et al., 1997
) and mast cells (Dvorak and Morgan, 2000
; Dvorak and Morgan, 2001
). In other organisms, similar observations have been made for the 3′ UTR of ASH1
mRNA in budding yeast (Hazelrigg, 1998
) and the bcd
mRNA in Drosophila
oocytes (Wang and Hazelrigg, 1994
; Oleynikov and Singer, 1998
; Bassell and Oleynikov, 1999
). In studies of fibroblasts, the β-actin mRNA is localized at the leading edge of the cell, and the 3′UTR binds a protein called zipcode-binding protein 1 (ZBP-1; Ross et al., 1997
). GW182 does not have significant sequence similarity to ZBP-1, but they both have a RRM and NLS in common. Although the putative NLS motif suggests that ZBP-1 and GW182 proteins may be able to enter the nucleus, both proteins are predominantly found in the cytoplasm. In the case of GW182, the evidence showed that cytoplasmic staining was observed using both the index human serum and the rabbit antibodies to the GW182 recombinant protein. In addition, the partial protein of GW182 tagged with GFP was localized to the cytoplasm, specifically to GWBs, and no expression was detected in the nucleus. Last, no differences were observed in the IIF staining pattern with formaldehyde or other fixation was used (our unpublished data compared methanol/acetone fixation with paraformaldehyde/Triton X-100 fixation). Interestingly, HuB, HuC, and HuD isoforms are mainly cytoplasmic in neurons with a small amount of protein observed with the nucleus as well (Gao and Keene, 1996
). Therefore, we believe that the potential NLS motif may either be nonfunctional or is blocked in cells grown under our conditions and requires a stimulus.
It is likely that GW182 protein is one member of a family of proteins residing in GWBs. As observed with many organelles and vesicles in the cell, it is simplistic to think that one protein comprises these complex structures. It is possible that more than one alternatively spliced protein product from the GW gene may reside in GWBs. First, the existence of two EST clones missing exon 10 (accession nos. BF169182 and W80996, from mouse and human, respectively) supports this possibility (Table ). Second, IP of extracts from radiolabeled HeLa cells by the index human serum reveals the presence of two proteins of 180 and 50 kDa. This could either be due to cross-reactivity or the association of these two proteins in a complex. We are currently pursuing isolating other proteins that are associated with GWBs.
Considering the sequence characteristics of GW182, it is not surprising that we were able to show that GW182 is likely a phosphoprotein. GWBs are heterogeneous in size and vary in number in individual cells. The variation in size and number may be related to the physiological state of the cell and the stage of the cell cycle. Detailed studies are currently underway to confirm this preliminary observation.
The significance of the GW repeats in GW182 is unclear. Although ESTs that contain this motif are in the database, to date no other mammalian proteins with this motif have been reported. A clue to the function of GW repeats may come from studies of bacteria such as Listeria monocytogenes
, Staphylococcus caprae,
and Erysipelothrix rhusiopathiae,
where it has been suggested that proteins bearing these repeats play an important role in anchoring bacterial proteins to the surface of the cell (Makino et al., 1998
) and anchoring bacteria to target cells (Braun et al., 1997
; Milohanic et al., 2001
). The mode of binding is not clearly understood, but is thought to occur via interaction with lipoteichoic acid or with specific cell surface proteins. For example, the protein internalin B (InIB) produced by L. monocytogenes
, is a key protein in promoting adherence and entry of the bacteria to host cells during infection. InIB has a C-terminal cell wall–anchoring domain containing 80-amino-acid GW repeats (Braun et al., 1997
). Deletion of this domain impaired adherence to host cells, whereas addition of GW repeats improved the binding to the cell surface. Similarly, the autolysin Ami in L. monocytogenes
contains a N-terminal catalytic domain and a C-terminal domain that is homologous to the GW domain in InIB but contains 8 GW modules arranged in tandem (Braun et al., 1997
; Milohanic et al., 2001
). Similar six to eight tandem repeat motifs have been described in the S. caprae atlC
gene product (Alligent et al., 2001
) and the surface protective antigen (SpaA) of E. rhusiopathiae
(Makino et al., 1998
). Of interest, the 6 GW repeats in S. caprae
occur in the fibronectin-binding domain of the atlC
protein. This may have relevance to the GW182 protein in mammalian cells that might bind to cytoskeletal elements to promote stabilization or movement of certain RNA species to their physiological target. The role of GW182 in binding or adherence of RNA or other proteins to cytoskeletal components or membrane moieties requires further study.