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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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Arginine methylation and SG dynamics. HeLa cells grown on cover slips were stressed with 0.5 mm sodium arsenite for 30 min. Cells were then returned to normal growth media and prepared for TDRD3/TIA-1 immunostaining after the indicated incubation periods (A). The experiment was repeated twice using different batches of cells. At least 500 cells were counted for each time point and condition. Fold increase of SG-positive cells in the MTA-treated cells when compared the DMSO-treated cells is depicted in a bar graph (B). Cell extracts prepared from HeLa cells grown in the presence (+) or the absence (−) of the general methylation inhibitor MTA were immunoblotted with the SYM10 antibody to confirm the reduction in steady-state arginine methylation levels. The same extracts were immunoblotted with actin antibodies to control for equal loading (C).
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DDN203F7: Arginine methylation and SG dynamics. HeLa cells grown on cover slips were stressed with 0.5 mm sodium arsenite for 30 min. Cells were then returned to normal growth media and prepared for TDRD3/TIA-1 immunostaining after the indicated incubation periods (A). The experiment was repeated twice using different batches of cells. At least 500 cells were counted for each time point and condition. Fold increase of SG-positive cells in the MTA-treated cells when compared the DMSO-treated cells is depicted in a bar graph (B). Cell extracts prepared from HeLa cells grown in the presence (+) or the absence (−) of the general methylation inhibitor MTA were immunoblotted with the SYM10 antibody to confirm the reduction in steady-state arginine methylation levels. The same extracts were immunoblotted with actin antibodies to control for equal loading (C).

Mentions: HeLa cells were grown for 20 h in the presence of 1 mm 5′-deoxy-5′-methylthioadenosine (MTA), a well-characterized general inhibitor of methylation (9). Following this pre-treatment, which reduced steady-state arginine methylation levels by ∼50% (Fig. 7C), cells were incubated with sodium arsenite and IF microscopy was performed with antibodies against TDRD3 and TIA-1 to monitor the formation of SGs. First, it was confirmed that the MTA treatment alone did not cause the formation of SGs (data not shown). As observed in non-treated HeLa cells (see time-course in Fig. 3D), TDRD3 was detectable in nascent SGs as early as 10–15 min after the addition of arsenite in mock-treated cells, and TIA-1 after ∼20 min (data not shown). SGs containing both TDRD3 and TIA-1 were present in 100% of cells after 30 min. Similarly, cells pre-treated with the methylation inhibitor MTA all contained TDRD3- and TIA-1-positive SGs after 30 min of stress stimulus, although small differences were observed at shorter time points. Specifically, recruitment of TDRD3 to SGs was delayed to some extent, while more cells with TIA-1-containing SGs could be clearly discerned after 15 min (data not shown). Thus, reducing arginine methylation levels by ∼50% did not prevent assembly of SGs, although the precise composition of these granules seemed to be altered.


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)

Arginine methylation and SG dynamics. HeLa cells grown on cover slips were stressed with 0.5 mm sodium arsenite for 30 min. Cells were then returned to normal growth media and prepared for TDRD3/TIA-1 immunostaining after the indicated incubation periods (A). The experiment was repeated twice using different batches of cells. At least 500 cells were counted for each time point and condition. Fold increase of SG-positive cells in the MTA-treated cells when compared the DMSO-treated cells is depicted in a bar graph (B). Cell extracts prepared from HeLa cells grown in the presence (+) or the absence (−) of the general methylation inhibitor MTA were immunoblotted with the SYM10 antibody to confirm the reduction in steady-state arginine methylation levels. The same extracts were immunoblotted with actin antibodies to control for equal loading (C).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2536506&req=5

DDN203F7: Arginine methylation and SG dynamics. HeLa cells grown on cover slips were stressed with 0.5 mm sodium arsenite for 30 min. Cells were then returned to normal growth media and prepared for TDRD3/TIA-1 immunostaining after the indicated incubation periods (A). The experiment was repeated twice using different batches of cells. At least 500 cells were counted for each time point and condition. Fold increase of SG-positive cells in the MTA-treated cells when compared the DMSO-treated cells is depicted in a bar graph (B). Cell extracts prepared from HeLa cells grown in the presence (+) or the absence (−) of the general methylation inhibitor MTA were immunoblotted with the SYM10 antibody to confirm the reduction in steady-state arginine methylation levels. The same extracts were immunoblotted with actin antibodies to control for equal loading (C).
Mentions: HeLa cells were grown for 20 h in the presence of 1 mm 5′-deoxy-5′-methylthioadenosine (MTA), a well-characterized general inhibitor of methylation (9). Following this pre-treatment, which reduced steady-state arginine methylation levels by ∼50% (Fig. 7C), cells were incubated with sodium arsenite and IF microscopy was performed with antibodies against TDRD3 and TIA-1 to monitor the formation of SGs. First, it was confirmed that the MTA treatment alone did not cause the formation of SGs (data not shown). As observed in non-treated HeLa cells (see time-course in Fig. 3D), TDRD3 was detectable in nascent SGs as early as 10–15 min after the addition of arsenite in mock-treated cells, and TIA-1 after ∼20 min (data not shown). SGs containing both TDRD3 and TIA-1 were present in 100% of cells after 30 min. Similarly, cells pre-treated with the methylation inhibitor MTA all contained TDRD3- and TIA-1-positive SGs after 30 min of stress stimulus, although small differences were observed at shorter time points. Specifically, recruitment of TDRD3 to SGs was delayed to some extent, while more cells with TIA-1-containing SGs could be clearly discerned after 15 min (data not shown). Thus, reducing arginine methylation levels by ∼50% did not prevent assembly of SGs, although the precise composition of these granules seemed to be altered.

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