TDRD3 is a multifunctional protein that acts as a transcriptional coactivator in the nucleus and as a scaffolding protein in cytoplasmic stress granules (Yang et al., 2010).

By searching databases with the tudor domain of SMN (see 600354), followed by PCR and 5-prime and 3-prime RACE of a brain cDNA library, Linder et al. (2008) cloned human TDRD3. The deduced 744-amino acid protein has an N-terminal oligosaccharide/nucleotide-binding (OB) fold, a central ubiquitin (191339)-associated (UBA) domain, and a C-terminal tudor domain. Northern blot analysis detected a 2.9-kb transcript variably expressed in all tissues examined. Immunofluorescence analysis of human cell lines revealed a strong cytosolic localization, with perinuclear accumulation and much weaker nuclear staining. Western blot analysis of HeLa and 293T cells showed that TDRD3 had an apparent molecular mass of about 83 kD.

Independently, Goulet et al. (2008) cloned and characterized TDRD3. Western blot analysis of fractionated HeLa cells detected TDRD3 at an apparent molecular mass of 83 kD predominantly in the cytosolic fraction, with much lower amounts in the nuclear and nuclear insoluble fractions.

Linder et al. (2008) found that the isolated UBA domain of TDRD3 bound to ubiquitin linked via lys48 in a tetraubiquitin chain, but not to monoubiquitin, lys63-linked ubiquitin, or SUMO1 (601912). Yeast 2-hybrid analysis of a human brain cDNA library revealed that TDRD3 interacted with FXR1 (600819). Protein pull-down and coimmunoprecipitation experiments showed that TDRD3 also interacted with FMRP (FMR1; 309550) and FXR2 (605339). Overexpression of TDRD3 or arsenite exposure in HeLa cells caused formation of stress granules containing TDRD3, FMRP, DAP5 (EIF4G2; 602325), and TIAR (TIAL1; 603413). Mutation analysis revealed that a region C-terminal to the tudor domain of TDRD3 interacted with 2 independent domains in FMRP. Linder et al. (2008) found that the ile304-to-asn (I304N; 309550.0001) mutation in FMRP, which is associated with fragile X syndrome (300624), abrogated the interaction between TDRD3 and FMRP.

Independently, Goulet et al. (2008) identified TDRD3 as a component of cytoplasmic stress granules. TDRD3 accumulated in stress granules upon arsenite-induced cell stress and colocalized with FMRP and the RNA-binding protein G3BP (608431). Both FMRP and TDRD3 followed polyribosomes isolated from both normal and stressed HeLa cells along a size-exclusion column. Mutation analysis revealed that the tudor domain of TDRD3 was necessary and sufficient for relocalization of TDRD3 to stress granules following arsenite treatment. Protein pull-down experiments showed that TDRD3 interacted with proteins involved in handling RNA, including the arginine- and glycine-rich proteins EWS (EWSR1; 133450), FUS (137070), DDX3 (DDX3X; 300160), SERBP1 (607378), and EEF1A1 (130590). Goulet et al. (2008) stated that the tudor domain is a symmetrical dimethylated arginine (sDMA)-binding motif, and they found that the isolated tudor domain of TDRD3 bound immobilized sDMA in vitro. Mutation of glu691 to lys (E691K) within the tudor domain of TDRD3 abrogated this interaction. The E691K mutation also eliminated or weakened interaction of TDRD3 with EWS, FUS, DDX3, and EEF1A1 and reduced recruitment of TDRD3 to stress granules.

Yang et al. (2010) screened a protein domain microarray for molecules that could read methylarginine marks associated with transcriptional activation. They found that TDRD3 bound to the activating asymmetric dimethylarginine mark on histone H4 (see 602822) arg3 (H4R3me2a) catalyzed by PRMT1 (602950) and to H3R17me2a (see 602810) catalyzed by CARM1 (603934). TDRD3 recognized the SDMA mark H4R3me2s, but more weakly. TDRD3 functioned as a coactivator in reporter assays in estrogen receptor (ER, or ESR1; 133430)- and androgen receptor (AR; 313700)-dependent reporter assays. Chromatin immunoprecipitation sequence analysis revealed that TDRD3 generally associated with transcriptional start sites and that it associated with the pS2 (TFF1; 113710) promoter in a CARM1-dependent manner in MCF7 human breast cancer cells. Knockdown of endogenous TDRD3 in MCF7 cells reduced ER-dependent reporter activity and reduced expression of TFF1, NRAS (164790), and DDX5 (180630). Yang et al. (2010) concluded that TDRD3 is a methylarginine-binding protein that has distinct roles in the nucleus and cytoplasm. They hypothesized that in the nucleus, TDRD3 is an effector molecule for CARM1- and PRMT1-generated methyl marks and functions as a transcriptional coactivator. In the cytoplasm, TDRD3 accumulates in stress granules in response to stress and functions as a scaffolding molecule.

Goulet et al. (2008) determined that the TDRD3 gene contains 14 exons and spans about 176.5 kb. Exon 14 is noncoding.

Goulet et al. (2008) stated that the TDRD3 gene maps to chromosome 13q21.2.