Limits...
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.

Show MeSH

Related in: MedlinePlus

Direct contact with methylated arginines contributes to TDRD3 relocalization to SGs. Biotinylated (RG4) peptides containing either unmethylated arginines or symmetrically dimethylated arginines (sDMA) were bound to streptavidin-agarose and used as affinity columns to measure the binding of purified GST fusion SMN and SMN E134K (as controls) and/or TDRD3 and TDRD3 E691K Tudor domains. The bound GST-Tdr fusion proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and detected by immunoblotting using GST antibodies (A). HeLa cells were transiently transfected with constructs expressing recombinant myc-tagged wild type or mutated (E691K) TDRD3 Tudor domain. 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 (B). HeLa cells were either left untreated (C) or treated with 0.5 mm sodium arsenite for 30 min (Ars). Cells were then labeled for immunofluorescence with EWS (a and b), DDX3 (c and d), FUS (e and f), or EEF1A1 (g and h) in combination with TIA-1 antibodies to detect endogenous proteins. HeLa cells transiently transfected to express myc-tagged SERBP1 (i and j) were either left untreated (C) or stressed as described above (Ars), before being immunostained with TDRD3 and myc (SERBP1) antibodies (C). HeLa cells from 2× 150 mm plates were lysed and incubated with purified recombinant GST (as a control), GST-TDRD3-Tdr or GST-TDRD3-Tdr E691K proteins coupled to glutathione-agarose. The retained proteins were resolved by SDS-PAGE, transferred to PVDF, and immunoblotted with the specified antibodies to confirm mass spectrometry identifications. The membrane was stained with Ponceau Red prior to immunoblotting, in order to show the GST-fusion proteins (D).
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2536506&req=5

DDN203F6: Direct contact with methylated arginines contributes to TDRD3 relocalization to SGs. Biotinylated (RG4) peptides containing either unmethylated arginines or symmetrically dimethylated arginines (sDMA) were bound to streptavidin-agarose and used as affinity columns to measure the binding of purified GST fusion SMN and SMN E134K (as controls) and/or TDRD3 and TDRD3 E691K Tudor domains. The bound GST-Tdr fusion proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and detected by immunoblotting using GST antibodies (A). HeLa cells were transiently transfected with constructs expressing recombinant myc-tagged wild type or mutated (E691K) TDRD3 Tudor domain. 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 (B). HeLa cells were either left untreated (C) or treated with 0.5 mm sodium arsenite for 30 min (Ars). Cells were then labeled for immunofluorescence with EWS (a and b), DDX3 (c and d), FUS (e and f), or EEF1A1 (g and h) in combination with TIA-1 antibodies to detect endogenous proteins. HeLa cells transiently transfected to express myc-tagged SERBP1 (i and j) were either left untreated (C) or stressed as described above (Ars), before being immunostained with TDRD3 and myc (SERBP1) antibodies (C). HeLa cells from 2× 150 mm plates were lysed and incubated with purified recombinant GST (as a control), GST-TDRD3-Tdr or GST-TDRD3-Tdr E691K proteins coupled to glutathione-agarose. The retained proteins were resolved by SDS-PAGE, transferred to PVDF, and immunoblotted with the specified antibodies to confirm mass spectrometry identifications. The membrane was stained with Ponceau Red prior to immunoblotting, in order to show the GST-fusion proteins (D).

Mentions: All the identified proteins also contain RG-rich motifs, which represent common target sites for protein arginine methyltransferases (Table 1). In fact, the presence of methylated arginines in EWS and FUS has been documented (6,7,72), and we have also detected modified arginines in our proteomic analysis (data not shown). Since the Tudor domain of TDRD3 can specifically recognize methylated arginine motifs in proteins (16,17), we speculated that TDRD3 might be recruited to SGs through methyl-dependent interactions with one or more of its binding partners. Previous studies have demonstrated that an E134K amino acid substitution in the Tudor domain of SMN abolished its interaction with arginine-methylated polypeptides (17). This amino acid is conserved in the Tudor domain of TDRD3 and corresponds to residue E691. In order to determine if the equivalent E691K substitution in TDRD3 Tudor domain would also abolish its capacity to interact with methylated arginines, an in vitro binding assay was used, with synthetic (RG)4 biotinylated peptides containing either no modified amino acids or symmetrically dimethylated arginines (sDMA). Purified recombinant GST-TDRD3-Tdr fusion proteins were incubated with the biotinylated peptides coupled to streptavidin-agarose. As previously reported (17), purified GST-TDRD3-Tdr bound to sDMA peptide columns in a dose-dependent manner, but did not bind the unmethylated peptide (Fig. 6A, compare lanes 5–7 with lanes 2–4). Remarkably, this binding was completely abolished with the introduction of the E691K mutation in the TDRD3 Tudor domain (Fig. 6A, GST-TDRD3-Tdr E691K). This result confirms that the Tudor domain of TDRD3 behaves as a methyl-binding protein module similar to that of SMN, in which the E691 residue is crucial for direct contact with dimethylated arginine motifs. This TDRD3 mutant was next used to determine if this methylarginine binding surface was important for recruitment to SGs. HeLa cells were transfected with a myc-tagged TDRD3 Tudor fragment containing the E691K point mutation. Twenty-four hours post-transfection, cells were stressed with sodium arsenite and labeled for IF with myc antibodies. Strikingly, the introduction of the E691K mutation in the Tudor domain of TDRD3 reduced its recruitment to SGs upon stress by at least 50% (Fig. 6B). This observation suggests that TDRD3's recruitment to SGs is likely mediated through specific protein–protein interactions involving methylated arginines.


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)

Direct contact with methylated arginines contributes to TDRD3 relocalization to SGs. Biotinylated (RG4) peptides containing either unmethylated arginines or symmetrically dimethylated arginines (sDMA) were bound to streptavidin-agarose and used as affinity columns to measure the binding of purified GST fusion SMN and SMN E134K (as controls) and/or TDRD3 and TDRD3 E691K Tudor domains. The bound GST-Tdr fusion proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and detected by immunoblotting using GST antibodies (A). HeLa cells were transiently transfected with constructs expressing recombinant myc-tagged wild type or mutated (E691K) TDRD3 Tudor domain. 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 (B). HeLa cells were either left untreated (C) or treated with 0.5 mm sodium arsenite for 30 min (Ars). Cells were then labeled for immunofluorescence with EWS (a and b), DDX3 (c and d), FUS (e and f), or EEF1A1 (g and h) in combination with TIA-1 antibodies to detect endogenous proteins. HeLa cells transiently transfected to express myc-tagged SERBP1 (i and j) were either left untreated (C) or stressed as described above (Ars), before being immunostained with TDRD3 and myc (SERBP1) antibodies (C). HeLa cells from 2× 150 mm plates were lysed and incubated with purified recombinant GST (as a control), GST-TDRD3-Tdr or GST-TDRD3-Tdr E691K proteins coupled to glutathione-agarose. The retained proteins were resolved by SDS-PAGE, transferred to PVDF, and immunoblotted with the specified antibodies to confirm mass spectrometry identifications. The membrane was stained with Ponceau Red prior to immunoblotting, in order to show the GST-fusion proteins (D).
© Copyright Policy
Related In: Results  -  Collection

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

DDN203F6: Direct contact with methylated arginines contributes to TDRD3 relocalization to SGs. Biotinylated (RG4) peptides containing either unmethylated arginines or symmetrically dimethylated arginines (sDMA) were bound to streptavidin-agarose and used as affinity columns to measure the binding of purified GST fusion SMN and SMN E134K (as controls) and/or TDRD3 and TDRD3 E691K Tudor domains. The bound GST-Tdr fusion proteins were resolved by SDS-PAGE, transferred to a PVDF membrane, and detected by immunoblotting using GST antibodies (A). HeLa cells were transiently transfected with constructs expressing recombinant myc-tagged wild type or mutated (E691K) TDRD3 Tudor domain. 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 (B). HeLa cells were either left untreated (C) or treated with 0.5 mm sodium arsenite for 30 min (Ars). Cells were then labeled for immunofluorescence with EWS (a and b), DDX3 (c and d), FUS (e and f), or EEF1A1 (g and h) in combination with TIA-1 antibodies to detect endogenous proteins. HeLa cells transiently transfected to express myc-tagged SERBP1 (i and j) were either left untreated (C) or stressed as described above (Ars), before being immunostained with TDRD3 and myc (SERBP1) antibodies (C). HeLa cells from 2× 150 mm plates were lysed and incubated with purified recombinant GST (as a control), GST-TDRD3-Tdr or GST-TDRD3-Tdr E691K proteins coupled to glutathione-agarose. The retained proteins were resolved by SDS-PAGE, transferred to PVDF, and immunoblotted with the specified antibodies to confirm mass spectrometry identifications. The membrane was stained with Ponceau Red prior to immunoblotting, in order to show the GST-fusion proteins (D).
Mentions: All the identified proteins also contain RG-rich motifs, which represent common target sites for protein arginine methyltransferases (Table 1). In fact, the presence of methylated arginines in EWS and FUS has been documented (6,7,72), and we have also detected modified arginines in our proteomic analysis (data not shown). Since the Tudor domain of TDRD3 can specifically recognize methylated arginine motifs in proteins (16,17), we speculated that TDRD3 might be recruited to SGs through methyl-dependent interactions with one or more of its binding partners. Previous studies have demonstrated that an E134K amino acid substitution in the Tudor domain of SMN abolished its interaction with arginine-methylated polypeptides (17). This amino acid is conserved in the Tudor domain of TDRD3 and corresponds to residue E691. In order to determine if the equivalent E691K substitution in TDRD3 Tudor domain would also abolish its capacity to interact with methylated arginines, an in vitro binding assay was used, with synthetic (RG)4 biotinylated peptides containing either no modified amino acids or symmetrically dimethylated arginines (sDMA). Purified recombinant GST-TDRD3-Tdr fusion proteins were incubated with the biotinylated peptides coupled to streptavidin-agarose. As previously reported (17), purified GST-TDRD3-Tdr bound to sDMA peptide columns in a dose-dependent manner, but did not bind the unmethylated peptide (Fig. 6A, compare lanes 5–7 with lanes 2–4). Remarkably, this binding was completely abolished with the introduction of the E691K mutation in the TDRD3 Tudor domain (Fig. 6A, GST-TDRD3-Tdr E691K). This result confirms that the Tudor domain of TDRD3 behaves as a methyl-binding protein module similar to that of SMN, in which the E691 residue is crucial for direct contact with dimethylated arginine motifs. This TDRD3 mutant was next used to determine if this methylarginine binding surface was important for recruitment to SGs. HeLa cells were transfected with a myc-tagged TDRD3 Tudor fragment containing the E691K point mutation. Twenty-four hours post-transfection, cells were stressed with sodium arsenite and labeled for IF with myc antibodies. Strikingly, the introduction of the E691K mutation in the Tudor domain of TDRD3 reduced its recruitment to SGs upon stress by at least 50% (Fig. 6B). This observation suggests that TDRD3's recruitment to SGs is likely mediated through specific protein–protein interactions involving methylated arginines.

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