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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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TDRD3 is associated with polyribosomes in HeLa cells. Cytoplasmic extracts were prepared from HeLa cells grown in normal conditions and centrifuged on a 10–60% w/w linear sucrose gradient. Fractions were collected and analyzed by western blot with antibodies against the ribosomal S6 protein and FMRP (as positive controls), TDRD3, and Hsp72 (as a negative control) (Control panel). Cytoplasmic extracts from HeLa cells treated with 0.5 mm sodium arsenite for 30 min were analyzed in parallel and the collected fractions were analyzed by western blot using the ribosomal L28 protein as positive control (Arsenite panel). Fractions from the top to the bottom of the gradient are shown from left to right. The positions of free small (40S) and large (60S) ribosomal subunits, monosomes (80S), and polysomes are indicated in each profile. The band corresponding to TDRD3 is indicated by a ‘dot’ on the side of the respective panels. Additional bands detected on the immunoblots represent non-specific reactivity with our polyclonal antibody.
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DDN203F4: TDRD3 is associated with polyribosomes in HeLa cells. Cytoplasmic extracts were prepared from HeLa cells grown in normal conditions and centrifuged on a 10–60% w/w linear sucrose gradient. Fractions were collected and analyzed by western blot with antibodies against the ribosomal S6 protein and FMRP (as positive controls), TDRD3, and Hsp72 (as a negative control) (Control panel). Cytoplasmic extracts from HeLa cells treated with 0.5 mm sodium arsenite for 30 min were analyzed in parallel and the collected fractions were analyzed by western blot using the ribosomal L28 protein as positive control (Arsenite panel). Fractions from the top to the bottom of the gradient are shown from left to right. The positions of free small (40S) and large (60S) ribosomal subunits, monosomes (80S), and polysomes are indicated in each profile. The band corresponding to TDRD3 is indicated by a ‘dot’ on the side of the respective panels. Additional bands detected on the immunoblots represent non-specific reactivity with our polyclonal antibody.

Mentions: SGs are considered to be stalled aggregates of 48S preinitiation complexes and contain a number of translation factors, including eIF3, eIF4E, eIF4G, PABP-1 and FMRP (50). In contrast to TIA-1, which is not normally associated with the translational machinery (63), FMRP can be found on polyribosomes (64,65). To determine if TDRD3 is associated with translational complexes, post-nuclear supernatants were subjected to velocity sedimentation through sucrose gradients and each collected fraction was analyzed by western blot for the presence of TDRD3 (Fig. 4, control panels). Using this protocol, it is possible to resolve free ribosomal subunits (40S and 60S) and monomers (80S) from actively translating polysomes, which sediment towards the bottom of the gradient, as confirmed by the distribution of the ribosomal S6 protein in each fraction (Fig. 4, S6 panel). Strikingly, TDRD3 was found distributed in both the light and the heavy sedimenting fractions of the sucrose gradient, following a pattern similar to what is observed for FMRP (Fig. 4, respective panels). Heat shock protein 72 (Hsp72) was used as a negative control in these experiments as it should not be associated with ribosomes. These results indicate that TDRD3 is associated with actively translating polyribosomes in cycling HeLa cells.


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 is associated with polyribosomes in HeLa cells. Cytoplasmic extracts were prepared from HeLa cells grown in normal conditions and centrifuged on a 10–60% w/w linear sucrose gradient. Fractions were collected and analyzed by western blot with antibodies against the ribosomal S6 protein and FMRP (as positive controls), TDRD3, and Hsp72 (as a negative control) (Control panel). Cytoplasmic extracts from HeLa cells treated with 0.5 mm sodium arsenite for 30 min were analyzed in parallel and the collected fractions were analyzed by western blot using the ribosomal L28 protein as positive control (Arsenite panel). Fractions from the top to the bottom of the gradient are shown from left to right. The positions of free small (40S) and large (60S) ribosomal subunits, monosomes (80S), and polysomes are indicated in each profile. The band corresponding to TDRD3 is indicated by a ‘dot’ on the side of the respective panels. Additional bands detected on the immunoblots represent non-specific reactivity with our polyclonal antibody.
© Copyright Policy
Related In: Results  -  Collection

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

DDN203F4: TDRD3 is associated with polyribosomes in HeLa cells. Cytoplasmic extracts were prepared from HeLa cells grown in normal conditions and centrifuged on a 10–60% w/w linear sucrose gradient. Fractions were collected and analyzed by western blot with antibodies against the ribosomal S6 protein and FMRP (as positive controls), TDRD3, and Hsp72 (as a negative control) (Control panel). Cytoplasmic extracts from HeLa cells treated with 0.5 mm sodium arsenite for 30 min were analyzed in parallel and the collected fractions were analyzed by western blot using the ribosomal L28 protein as positive control (Arsenite panel). Fractions from the top to the bottom of the gradient are shown from left to right. The positions of free small (40S) and large (60S) ribosomal subunits, monosomes (80S), and polysomes are indicated in each profile. The band corresponding to TDRD3 is indicated by a ‘dot’ on the side of the respective panels. Additional bands detected on the immunoblots represent non-specific reactivity with our polyclonal antibody.
Mentions: SGs are considered to be stalled aggregates of 48S preinitiation complexes and contain a number of translation factors, including eIF3, eIF4E, eIF4G, PABP-1 and FMRP (50). In contrast to TIA-1, which is not normally associated with the translational machinery (63), FMRP can be found on polyribosomes (64,65). To determine if TDRD3 is associated with translational complexes, post-nuclear supernatants were subjected to velocity sedimentation through sucrose gradients and each collected fraction was analyzed by western blot for the presence of TDRD3 (Fig. 4, control panels). Using this protocol, it is possible to resolve free ribosomal subunits (40S and 60S) and monomers (80S) from actively translating polysomes, which sediment towards the bottom of the gradient, as confirmed by the distribution of the ribosomal S6 protein in each fraction (Fig. 4, S6 panel). Strikingly, TDRD3 was found distributed in both the light and the heavy sedimenting fractions of the sucrose gradient, following a pattern similar to what is observed for FMRP (Fig. 4, respective panels). Heat shock protein 72 (Hsp72) was used as a negative control in these experiments as it should not be associated with ribosomes. These results indicate that TDRD3 is associated with actively translating polyribosomes in cycling HeLa cells.

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