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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.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.

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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The Tudor domain of TDRD3 is both required and sufficient for its recruitment to stress granules. Diagram showing the various myc epitope-tagged TDRD3 deletion mutants used in this study (A). HeLa cells were transiently transfected with each deletion mutant. Twenty-four hours post-transfection, cells were left untreated and labeled for immunofluorescence with TIA-1 and myc antibodies to detect endogenous TIA-1 protein and recombinant myc-tagged proteins (B). Alternatively, transfected cells were treated with 0.5 mm sodium arsenite for 30 min. Indirect immunofluorescence staining was performed as described above (C). HeLa cells were transiently transfected with constructs expressing recombinant myc-tagged Tudor domain of SMN, SPF30 or TDRD3. Twenty-four hours post-transfection, cells were treated with 0.5 mm sodium arsenite for 30 min. Indirect immunofluorescence staining was performed using myc and TIA-1 antibodies (D). Total cell lysates of HeLa cells transiently expressing myc-tagged TDRD3 deletion mutants, as well as SMN and SPF30 Tudor domains, were immunoblotted with myc antibodies to confirm expression (E).
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DDN203F5: The Tudor domain of TDRD3 is both required and sufficient for its recruitment to stress granules. Diagram showing the various myc epitope-tagged TDRD3 deletion mutants used in this study (A). HeLa cells were transiently transfected with each deletion mutant. Twenty-four hours post-transfection, cells were left untreated and labeled for immunofluorescence with TIA-1 and myc antibodies to detect endogenous TIA-1 protein and recombinant myc-tagged proteins (B). Alternatively, transfected cells were treated with 0.5 mm sodium arsenite for 30 min. Indirect immunofluorescence staining was performed as described above (C). HeLa cells were transiently transfected with constructs expressing recombinant myc-tagged Tudor domain of SMN, SPF30 or TDRD3. Twenty-four hours post-transfection, cells were treated with 0.5 mm sodium arsenite for 30 min. Indirect immunofluorescence staining was performed using myc and TIA-1 antibodies (D). Total cell lysates of HeLa cells transiently expressing myc-tagged TDRD3 deletion mutants, as well as SMN and SPF30 Tudor domains, were immunoblotted with myc antibodies to confirm expression (E).

Mentions: In order to characterize the TDRD3 protein, a rabbit polyclonal antibody was raised against a synthetic peptide [coupled to keyhole limpet hemocyanine (KLH)] corresponding to the last 22 C-terminal amino acids of TDRD3 (723DGQPRRSTRPTQQFYQPPRARN744). The affinity-purified antibody recognized a single band of ∼83 kDa on immunoblots (IB) performed using HeLa cell extracts (Fig. 2A, lane 1), which corresponds to the predicted molecular weight of TDRD3. Immunoreactivity to endogenous TDRD3 in western blots was completely abolished by preincubation of TDRD3 antibodies with the antigenic peptide, but not with an unrelated peptide, demonstrating the specificity of our antibody (Fig. 2A, compare lanes 2 with 3). To further confirm that our antibodies specifically recognize the TDRD3 protein, in vitro coupled transcription/translation (IVTT) reactions were programmed with a construct designed to produce a hexahistidine-tagged truncated version of TDRD3 (ΔN328 in Fig. 5A), which still contains the reactive epitope. This TDRD3 C-terminal domain was effectively recognized by our TDRD3 antiserum in western blot analysis of the programmed IVTT reactions but not in control reactions (Fig. 2B, compare lanes 2 with 1), confirming that our antibody recognizes the TDRD3 protein sequence. Interestingly, the antibody also specifically recognized a single band corresponding to TDRD3 predicted molecular weight in the rabbit reticulocyte lysates, suggesting that TDRD3 is found endogenously in these lysates (Fig. 2B, lanes 1 and 2).


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)

The Tudor domain of TDRD3 is both required and sufficient for its recruitment to stress granules. Diagram showing the various myc epitope-tagged TDRD3 deletion mutants used in this study (A). HeLa cells were transiently transfected with each deletion mutant. Twenty-four hours post-transfection, cells were left untreated and labeled for immunofluorescence with TIA-1 and myc antibodies to detect endogenous TIA-1 protein and recombinant myc-tagged proteins (B). Alternatively, transfected cells were treated with 0.5 mm sodium arsenite for 30 min. Indirect immunofluorescence staining was performed as described above (C). HeLa cells were transiently transfected with constructs expressing recombinant myc-tagged Tudor domain of SMN, SPF30 or TDRD3. Twenty-four hours post-transfection, cells were treated with 0.5 mm sodium arsenite for 30 min. Indirect immunofluorescence staining was performed using myc and TIA-1 antibodies (D). Total cell lysates of HeLa cells transiently expressing myc-tagged TDRD3 deletion mutants, as well as SMN and SPF30 Tudor domains, were immunoblotted with myc antibodies to confirm expression (E).
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2536506&req=5

DDN203F5: The Tudor domain of TDRD3 is both required and sufficient for its recruitment to stress granules. Diagram showing the various myc epitope-tagged TDRD3 deletion mutants used in this study (A). HeLa cells were transiently transfected with each deletion mutant. Twenty-four hours post-transfection, cells were left untreated and labeled for immunofluorescence with TIA-1 and myc antibodies to detect endogenous TIA-1 protein and recombinant myc-tagged proteins (B). Alternatively, transfected cells were treated with 0.5 mm sodium arsenite for 30 min. Indirect immunofluorescence staining was performed as described above (C). HeLa cells were transiently transfected with constructs expressing recombinant myc-tagged Tudor domain of SMN, SPF30 or TDRD3. Twenty-four hours post-transfection, cells were treated with 0.5 mm sodium arsenite for 30 min. Indirect immunofluorescence staining was performed using myc and TIA-1 antibodies (D). Total cell lysates of HeLa cells transiently expressing myc-tagged TDRD3 deletion mutants, as well as SMN and SPF30 Tudor domains, were immunoblotted with myc antibodies to confirm expression (E).
Mentions: In order to characterize the TDRD3 protein, a rabbit polyclonal antibody was raised against a synthetic peptide [coupled to keyhole limpet hemocyanine (KLH)] corresponding to the last 22 C-terminal amino acids of TDRD3 (723DGQPRRSTRPTQQFYQPPRARN744). The affinity-purified antibody recognized a single band of ∼83 kDa on immunoblots (IB) performed using HeLa cell extracts (Fig. 2A, lane 1), which corresponds to the predicted molecular weight of TDRD3. Immunoreactivity to endogenous TDRD3 in western blots was completely abolished by preincubation of TDRD3 antibodies with the antigenic peptide, but not with an unrelated peptide, demonstrating the specificity of our antibody (Fig. 2A, compare lanes 2 with 3). To further confirm that our antibodies specifically recognize the TDRD3 protein, in vitro coupled transcription/translation (IVTT) reactions were programmed with a construct designed to produce a hexahistidine-tagged truncated version of TDRD3 (ΔN328 in Fig. 5A), which still contains the reactive epitope. This TDRD3 C-terminal domain was effectively recognized by our TDRD3 antiserum in western blot analysis of the programmed IVTT reactions but not in control reactions (Fig. 2B, compare lanes 2 with 1), confirming that our antibody recognizes the TDRD3 protein sequence. Interestingly, the antibody also specifically recognized a single band corresponding to TDRD3 predicted molecular weight in the rabbit reticulocyte lysates, suggesting that TDRD3 is found endogenously in these lysates (Fig. 2B, lanes 1 and 2).

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.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.

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