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TDRD3, a novel Tudor domain-containing protein, localizes to cytoplasmic stress granules.

Goulet I, Boisvenue S, Mokas S, Mazroui R, Côté J - Hum. Mol. Genet. (2008)

Bottom Line: TDRD3 is a modular protein, and in addition to its Tudor domain, it harbors a putative nucleic acid recognition motif and a ubiquitin-associated domain.Strikingly, the Tudor domain of TDRD3 was found to be both required and sufficient for its recruitment to SGs, and the methyl-binding surface in the Tudor domain is important for this process.Taken together, we report the first characterization of TDRD3 and its functional interaction with at least two proteins implicated in human genetic diseases and present evidence supporting a role for arginine methylation in the regulation of SG dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine and Centre for Neuromuscular Disease, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5.

ABSTRACT
Our previous work has demonstrated that the Tudor domain of the 'survival of motor neuron' protein and the Tudor domain-containing protein 3 (TDRD3) are highly similar and that they both have the ability to interact with arginine-methylated polypeptides. TDRD3 has been identified among genes whose overexpression has a strong predictive value for poor prognosis of estrogen receptor-negative breast cancers, although its precise function remains unknown. TDRD3 is a modular protein, and in addition to its Tudor domain, it harbors a putative nucleic acid recognition motif and a ubiquitin-associated domain. We report here that TDRD3 localizes predominantly to the cytoplasm, where it co-sediments with the fragile X mental retardation protein on actively translating polyribosomes. We also demonstrate that TDRD3 accumulates into stress granules (SGs) in response to various cellular stresses. Strikingly, the Tudor domain of TDRD3 was found to be both required and sufficient for its recruitment to SGs, and the methyl-binding surface in the Tudor domain is important for this process. Pull down experiments identified five novel TDRD3 interacting partners, most of which are potentially methylated RNA-binding proteins. Our findings revealed that two of these proteins, SERPINE1 mRNA-binding protein 1 and DEAD/H box-3 (a gene often deleted in Sertoli-cell-only syndrome), are also novel constituents of cytoplasmic SGs. Taken together, we report the first characterization of TDRD3 and its functional interaction with at least two proteins implicated in human genetic diseases and present evidence supporting a role for arginine methylation in the regulation of SG dynamics.

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TDRD3 localizes to cytoplasmic stress granules during stress response. HeLa cells cultured on glass cover slips were left untreated (C) or exposed to oxidative stress (Ars; 0.5 mm sodium arsenite for 30 min), heat shock (HS; 43°C for 30 min) or high-osmolarity medium (Osm; 1 M sorbitol in DMEM for 1 h followed by a 30-min recovery in normal DMEM). The cells were fixed and immunostained with TDRD3 and TIA-1 antibodies to detect the endogenous proteins (A). Cells were stressed with 0.5 mm sodium arsenite (Ars) for 30 min, followed by immunostaining of both TDRD3 and FMRP endogenous proteins (B). To visualize G3BP, a GFP fusion construct was transfected into cells 24 h prior to immunofluorescence analysis. Cells were left untreated (C) or exposed to oxidative stress (Ars) for 30 min, followed by immunostaining for endogenous TDRD3 (C). Cells were stressed with 0.5 mm sodium arsenite for the indicated time length before their fixation and double immunofluorescence with TDRD3 and TIA-1 antibodies (D). HeLa cells were either left untreated (C) or stressed for 30 min with 0.5 mm sodium arsenite (Ars). After fixation, endogenous TDRD3 and GW182 (a marker of P-bodies) were detected by fluorescence microscopy (E).
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DDN203F3: TDRD3 localizes to cytoplasmic stress granules during stress response. HeLa cells cultured on glass cover slips were left untreated (C) or exposed to oxidative stress (Ars; 0.5 mm sodium arsenite for 30 min), heat shock (HS; 43°C for 30 min) or high-osmolarity medium (Osm; 1 M sorbitol in DMEM for 1 h followed by a 30-min recovery in normal DMEM). The cells were fixed and immunostained with TDRD3 and TIA-1 antibodies to detect the endogenous proteins (A). Cells were stressed with 0.5 mm sodium arsenite (Ars) for 30 min, followed by immunostaining of both TDRD3 and FMRP endogenous proteins (B). To visualize G3BP, a GFP fusion construct was transfected into cells 24 h prior to immunofluorescence analysis. Cells were left untreated (C) or exposed to oxidative stress (Ars) for 30 min, followed by immunostaining for endogenous TDRD3 (C). Cells were stressed with 0.5 mm sodium arsenite for the indicated time length before their fixation and double immunofluorescence with TDRD3 and TIA-1 antibodies (D). HeLa cells were either left untreated (C) or stressed for 30 min with 0.5 mm sodium arsenite (Ars). After fixation, endogenous TDRD3 and GW182 (a marker of P-bodies) were detected by fluorescence microscopy (E).

Mentions: In order to confirm the identity of these TDRD3-containing foci, HeLa cells were exposed to different stress stimuli and double IF experiments were performed using TDRD3 antibodies in combination with an antibody directed against the RNA-binding protein TIA-1, a well-accepted marker of cytoplasmic SGs. Upon oxidative, heat and osmotic shocks, endogenous TDRD3 was redistributed to cytoplasmic SGs, where it colocalized with TIA-1 (Fig. 3A). For simplicity, sodium arsenite was used in all subsequent experiments to induce cellular stress response. The FMRP is another protein that is well-known to relocalize to SGs (57,58), and it was recently found to interact with TDRD3 (U. Fischer, personal communication). Accordingly, colocalization of TDRD3 and FMRP into SGs following stress was also observed (Fig. 3B). Relocalization of TDRD3 in SGs was further confirmed by its colocalization with a GFP fusion of another component of these granules, the RNA-binding protein G3BP (Fig. 3C, bottom, Ars). G3BP overexpression is known to induce the formation of SGs, even in the absence of any other stress stimuli (59), and as anticipated, TDRD3 was also observed in G3BP-induced SGs (Fig. 3C, top, C). Importantly, TDRD3 was detectable in SGs as early as 10 min following sodium arsenite treatment (Fig. 3D), which is comparable to the kinetics of TIA-1 relocalization to SGs (60). However, the use of finer live cell imaging approaches will be required to determine the precise kinetics of TDRD3 recruitment to SGs. Finally, TDRD3 did not localize to other cytoplasmic mRNA foci, such as processing bodies, as shown by the absence of colocalization with GW182 (Fig. 3E), an RNA-binding protein resident of these structures (61,62). Taken together, our results indicate that TDRD3 is a novel component of cytoplasmic SGs.


TDRD3, a novel Tudor domain-containing protein, localizes to cytoplasmic stress granules.

Goulet I, Boisvenue S, Mokas S, Mazroui R, Côté J - Hum. Mol. Genet. (2008)

TDRD3 localizes to cytoplasmic stress granules during stress response. HeLa cells cultured on glass cover slips were left untreated (C) or exposed to oxidative stress (Ars; 0.5 mm sodium arsenite for 30 min), heat shock (HS; 43°C for 30 min) or high-osmolarity medium (Osm; 1 M sorbitol in DMEM for 1 h followed by a 30-min recovery in normal DMEM). The cells were fixed and immunostained with TDRD3 and TIA-1 antibodies to detect the endogenous proteins (A). Cells were stressed with 0.5 mm sodium arsenite (Ars) for 30 min, followed by immunostaining of both TDRD3 and FMRP endogenous proteins (B). To visualize G3BP, a GFP fusion construct was transfected into cells 24 h prior to immunofluorescence analysis. Cells were left untreated (C) or exposed to oxidative stress (Ars) for 30 min, followed by immunostaining for endogenous TDRD3 (C). Cells were stressed with 0.5 mm sodium arsenite for the indicated time length before their fixation and double immunofluorescence with TDRD3 and TIA-1 antibodies (D). HeLa cells were either left untreated (C) or stressed for 30 min with 0.5 mm sodium arsenite (Ars). After fixation, endogenous TDRD3 and GW182 (a marker of P-bodies) were detected by fluorescence microscopy (E).
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DDN203F3: TDRD3 localizes to cytoplasmic stress granules during stress response. HeLa cells cultured on glass cover slips were left untreated (C) or exposed to oxidative stress (Ars; 0.5 mm sodium arsenite for 30 min), heat shock (HS; 43°C for 30 min) or high-osmolarity medium (Osm; 1 M sorbitol in DMEM for 1 h followed by a 30-min recovery in normal DMEM). The cells were fixed and immunostained with TDRD3 and TIA-1 antibodies to detect the endogenous proteins (A). Cells were stressed with 0.5 mm sodium arsenite (Ars) for 30 min, followed by immunostaining of both TDRD3 and FMRP endogenous proteins (B). To visualize G3BP, a GFP fusion construct was transfected into cells 24 h prior to immunofluorescence analysis. Cells were left untreated (C) or exposed to oxidative stress (Ars) for 30 min, followed by immunostaining for endogenous TDRD3 (C). Cells were stressed with 0.5 mm sodium arsenite for the indicated time length before their fixation and double immunofluorescence with TDRD3 and TIA-1 antibodies (D). HeLa cells were either left untreated (C) or stressed for 30 min with 0.5 mm sodium arsenite (Ars). After fixation, endogenous TDRD3 and GW182 (a marker of P-bodies) were detected by fluorescence microscopy (E).
Mentions: In order to confirm the identity of these TDRD3-containing foci, HeLa cells were exposed to different stress stimuli and double IF experiments were performed using TDRD3 antibodies in combination with an antibody directed against the RNA-binding protein TIA-1, a well-accepted marker of cytoplasmic SGs. Upon oxidative, heat and osmotic shocks, endogenous TDRD3 was redistributed to cytoplasmic SGs, where it colocalized with TIA-1 (Fig. 3A). For simplicity, sodium arsenite was used in all subsequent experiments to induce cellular stress response. The FMRP is another protein that is well-known to relocalize to SGs (57,58), and it was recently found to interact with TDRD3 (U. Fischer, personal communication). Accordingly, colocalization of TDRD3 and FMRP into SGs following stress was also observed (Fig. 3B). Relocalization of TDRD3 in SGs was further confirmed by its colocalization with a GFP fusion of another component of these granules, the RNA-binding protein G3BP (Fig. 3C, bottom, Ars). G3BP overexpression is known to induce the formation of SGs, even in the absence of any other stress stimuli (59), and as anticipated, TDRD3 was also observed in G3BP-induced SGs (Fig. 3C, top, C). Importantly, TDRD3 was detectable in SGs as early as 10 min following sodium arsenite treatment (Fig. 3D), which is comparable to the kinetics of TIA-1 relocalization to SGs (60). However, the use of finer live cell imaging approaches will be required to determine the precise kinetics of TDRD3 recruitment to SGs. Finally, TDRD3 did not localize to other cytoplasmic mRNA foci, such as processing bodies, as shown by the absence of colocalization with GW182 (Fig. 3E), an RNA-binding protein resident of these structures (61,62). Taken together, our results indicate that TDRD3 is a novel component of cytoplasmic SGs.

Bottom Line: TDRD3 is a modular protein, and in addition to its Tudor domain, it harbors a putative nucleic acid recognition motif and a ubiquitin-associated domain.Strikingly, the Tudor domain of TDRD3 was found to be both required and sufficient for its recruitment to SGs, and the methyl-binding surface in the Tudor domain is important for this process.Taken together, we report the first characterization of TDRD3 and its functional interaction with at least two proteins implicated in human genetic diseases and present evidence supporting a role for arginine methylation in the regulation of SG dynamics.

View Article: PubMed Central - PubMed

Affiliation: Department of Cellular and Molecular Medicine and Centre for Neuromuscular Disease, Faculty of Medicine, University of Ottawa, Ottawa, ON, Canada K1H 8M5.

ABSTRACT
Our previous work has demonstrated that the Tudor domain of the 'survival of motor neuron' protein and the Tudor domain-containing protein 3 (TDRD3) are highly similar and that they both have the ability to interact with arginine-methylated polypeptides. TDRD3 has been identified among genes whose overexpression has a strong predictive value for poor prognosis of estrogen receptor-negative breast cancers, although its precise function remains unknown. TDRD3 is a modular protein, and in addition to its Tudor domain, it harbors a putative nucleic acid recognition motif and a ubiquitin-associated domain. We report here that TDRD3 localizes predominantly to the cytoplasm, where it co-sediments with the fragile X mental retardation protein on actively translating polyribosomes. We also demonstrate that TDRD3 accumulates into stress granules (SGs) in response to various cellular stresses. Strikingly, the Tudor domain of TDRD3 was found to be both required and sufficient for its recruitment to SGs, and the methyl-binding surface in the Tudor domain is important for this process. Pull down experiments identified five novel TDRD3 interacting partners, most of which are potentially methylated RNA-binding proteins. Our findings revealed that two of these proteins, SERPINE1 mRNA-binding protein 1 and DEAD/H box-3 (a gene often deleted in Sertoli-cell-only syndrome), are also novel constituents of cytoplasmic SGs. Taken together, we report the first characterization of TDRD3 and its functional interaction with at least two proteins implicated in human genetic diseases and present evidence supporting a role for arginine methylation in the regulation of SG dynamics.

Show MeSH
Related in: MedlinePlus