Trax (translin-associated factor X), a primarily cytoplasmic protein, inhibits the binding of TB-RBP (translin) to RNA

Trax (Translin-associated factor X) has been shown to interact with TB-RBP/Translin by its coimmunoprecipitation and in yeast two-hybrid assays. Here we demonstrate that Trax is widely expressed, does not bind to DNA or RNA, but forms heterodimers with TB-RBP under reducing conditions. The heterodimer of TB-RBP and Trax inhibits TB-RBP binding to RNA, but enhances TB-RBP binding to specific single stranded DNA sequences. The in vitro interactions between TB-RBP and Trax are confirmed by similar interactions in the yeast two-hybrid system. Cell fractionation and confocal microscope studies reveal that Trax is predominantly cytoplasmic. In contrast, TB-RBP is present in both the nuclei and cytoplasm of transfected cells and uses a highly conserved nuclear export signal to exit nuclei. In addition to a leucine zipper, two basic domains in TB-RBP are essential for RNA binding, but only one of these domains is needed for DNA binding. Trax restores DNA binding to TB-RBP containing an altered form of this domain. These data suggest that Trax-TB.RBP interactions modulate the DNA- and RNA-binding activity of TB-RBP.     

Altering the GTP binding site of the DNA/RNA-binding protein, Translin/TB-RBP, decreases RNA binding and may create a dominant negative phenotype

The DNA/RNA-binding protein, Translin/Testis Brain RNA-binding protein (Translin/TB-RBP), contains a putative GTP binding site in its C-terminus which is highly conserved. To determine if guanine nucleotide binding to this site functionally alters nucleic acid binding, electrophoretic mobility shift assays were performed with RNA and DNA binding probes. GTP, but not GDP, reduces RNA binding by ~50% and the poorly hydrolyzed GTP analog, GTPγS, reduces binding by >90% in gel shift and immunoprecipitation assays. No similar reduction of DNA binding is seen. When the putative GTP binding site of TB-RBP, amino acid sequence VTAGD, is altered to VTNSD by site directed mutagenesis, GTP will no longer bind to TB-RBP_GTP and TB-RBP_GTP no longer binds to RNA, although DNA binding is not affected. Yeast two-hybrid assays reveal that like wild-type TB-RBP, TB-RBP_GTP will interact with itself, with wild-type TB-RBP and with Translin associated factor X (Trax). Transfection of TB-RBP_GTP into NIH 3T3 cells leads to a marked increase in cell death suggesting a dominant negative function for TB-RBP_GTP in cells. These data suggest TB-RBP is an RNA-binding protein whose activity is allosterically controlled by nucleotide binding.     

Mouse testis brain ribonucleic acid-binding protein/translin colocalizes with microtubules and is immunoprecipitated with messenger ribonucleic acids encoding myelin basic protein, alpha calmodulin kinase II, and protamines 1 and 2

Testis brain RNA-binding protein (TB-RBP) is a sequence-dependent RNA-binding protein that binds to conserved Y and H sequence elements present in many brain and testis mRNAs. Using recombinant TB-RBP and a highly enriched tubulin fraction, we demonstrate here that recombinant TB-RBP binds to microtubules assembled in vitro. The interaction between recombinant TB-RBP and microtubules was inhibited by high salt and by the microtubule disassembling agents colcemid and calcium, but not by the microfilament-disassembling agent cytochalasin D. Confocal microscopy confirmed colocalization of TB-RBP and tubulin in the cytoplasm of male germ cells. An affinity-purified antibody prepared against recombinant TB-RBP specifically precipitated mRNAs encoding myelin basic protein and alpha calmodulin-dependent kinase II-two transported mRNAs, and protamines 1 and 2-two translationally regulated testicular mRNAs. These data indicate an intracellular association between TB-RBP and specific target mRNAs and suggest an involvement of TB-RBP in microtubule-dependent mRNA transport in the cytoplasm of cells.     

Part of Xenopus translin is localized in the centrosomes during mitosis

During oogenesis, maternal mRNAs are synthesised and stored in a translationally dormant form due to the presence of regulatory elements at the 3' untranslated regions (3'UTR). In Xenopus oocytes, several studies have described the presence of RNA-binding proteins capable to repress maternal-mRNA translation. The testis-brain RNA-binding protein (TB-RBP/Translin) is a single-stranded DNA- and RNA-binding protein which can bind the 3' UTR regions (Y and H elements) of stored mRNAs and can suppress in vitro translation of the mRNAs that contain these sequences. Here we report the cloning of the Xenopus homologue of the TB-RBP/Translin protein (X-translin) as well as its expression, its localisation, and its biochemical association with the protein named Translin associated factor X (Trax) in Xenopus oocytes. The fact that this protein is highly present in the cytoplasm from stage VI oocytes until 48 h embryos and that it has been described as capable to inhibit paternal mRNA translation, indicates that it could play an important role in maternal mRNA translation control during Xenopus oogenesis and embryogenesis. Moreover, we investigated X-translin localisation during cell cycle in XTC cells. In interphase, although a weak and diffuse nuclear staining was observed, X-translin was mostly present in the cytoplasm where it exhibited a prominent granular staining. Interestingly, part of X-translin underwent a remarkable redistribution throughout mitosis and associated with centrosomes, which may suggest a new unknown role for this protein in cell cycle.     

Crystal structure of TB-RBP,a novel RNA-binding and regulating protein

The testis/brain-RNA-binding protein (TB-RBP) spatially and temporally controls the expression of specific mRNAs in developing male germ cells and brain cells, and is implicated in DNA recombination and repair events. We report the 2.65 A crystal structure of mouse TB-RBP. The structure is predominantly alpha-helical and exhibits a novel protein fold and mode of assembly. Crystal symmetry and molecular symmetry combine to form an octet of TB-RBP monomers in the shape of an elongated spherical particle with a large cavity at its center. Amino acid residues that affect RNA and DNA binding are located on the interior surface of the assembled particle, and a putative nucleotide-binding domain that controls RNA binding is located at a dimer interface. Other modes of assembly are suggested for TB-RBP based on our structure and recently reported electron microscopic reconstructions of human TB-RBP.     

Molecular model for DNA recognition by the family of basic-helix-loop-helix-zipper proteins.

The basic-helix-loop-helix-zipper (bHLH-Zip) motif is a conserved region of approximately 70 amino acids that mediates both sequence-specific DNA binding and protein dimerization. This motif is found in protein sequences from many eukaryotic organisms and is contained in the protein sequence of the oncogene myc and its partner max, and a shortened version of the motif (bHLH) is found in the muscle determination factor myoD and its partner E12. An evaluation of the conserved amino acids that define the motif coupled with the published mutagenic studies of this region has led to our formulation of a molecular model for the binding of this motif as a dimer to specific sequences of DNA. This model has the dimeric protein interacting with an abutted, dyad-symmetric DNA sequence. Helix 2 of each monomer is modeled as a coiled-coil extension of the C-terminal "leucine zipper." Helix 1 does not interact with helix 1 from its partner in the dimer but with the hydrophobic surface created when the helix 2 regions of the dimer interact with each other as a coiled-coil. Sequence-specific interactions are proposed between the basic region and the invariant cis elements that all bHLH-Zip proteins bind.     

Leucine zipper motif of chicken histone acetyltransferase-1 is essential for in vivo and in vitro interactions with the p48 subunit of chicken chromatin assembly factor-1

We cloned cDNA encoding chicken cytoplasmic histone acetyltransferase-1, chHAT-1, comprising 408 amino acids including a putative initiation Met. It exhibits 80.4% identity to the human homolog and possesses a typical leucine zipper motif. The glutathione S-transferase (GST) pull-down assay, involving truncated and missense mutants of the chicken chromatin assembly factor-1 (chCAF-1)p48, revealed not only that a region (comprising amino acids 376–405 of chCAF-1p48 and containing the seventh WD dipeptide motif) binds to chHAT-1 in vitro, but also that mutation of the motif has no influence on the in vitro interaction. The GST pull-down assay, involving truncated and missense chHAT-1 mutants, established that a region, comprising amino acids 380–408 of chHAT-1 and containing the leucine zipper motif, is required for its in vitro interaction with chCAF-1p48. In addition, mutation of each of four Leu residues in the leucine zipper motif prevents the in vitro interaction. The yeast two-hybrid assay revealed that all four Leu residues within the leucine zipper motif of chHAT-1 are necessary for its in vivo interaction with chCAF-1p48. These results indicate not only that the proper leucine zipper motif of chHAT-1 is essential for its interaction with chCAF-1p48, but also that the propeller structure of chCAF-1p48 expected to act as a platform for protein–protein interactions may not be necessary for this interaction of chHAT-1.     

Protein-protein interactions between the testis brain RNA-binding protein and the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin and Trax in male germ cells and the brain.

Numerous functions have been proposed for the testis brain RNA-binding protein (TB-RBP) and its human homologue, Translin, ranging from mRNA transport and translational regulation to DNA rearrangement and repair. To gain insight into the likely functions of this 26 kDa protein, immunoprecipitation was used to identify proteins that interact with TB-RBP in mouse cytosolic extracts. Three proteins, the transitional endoplasmic reticulum ATPase, a cytoskeletal gamma actin, and Trax, were specifically immunoprecipitated with an affinity-purified antibody to recombinant mouse TB-RBP. In vitro binding assays with recombinant proteins and EM immunocytochemistry confirm that TB-RBP interacts with the TER ATPase in vitro and in vivo. Confocal microscopy has demonstrated that TB-RBP colocalizes with actin in the cytoplasm of male germ cells. The immunoprecipitation of Trax with TB-RBP confirms a published report demonstrating protein interactions between the two proteins in a yeast two-hybrid assay. These data support the hypothesis that TB-RBP serves as a link in attaching specific mRNAs to cytoskeletal structures and suggests an involvement for the ubiquitously expressed TER ATPase in intracellular and/or intercellular mRNA transport.     

AU-rich RNA-binding induces changes in the quaternary structure of AUH

The human AU RNA binding protein/enoyl-Coenzyme A hydratase (AUH) is a 3-hydroxy-3-methylglutaconyl-CoA dehydratase in the leucine degradation pathway. It also possesses an RNA-binding activity to AUUU repeats, which involves no known conserved RNA-binding domains and is seemingly unrelated to the enzymatic activity. In this study, we performed mass spectrometric analyses to elucidate the oligomeric states of AUH in the presence and absence of RNA. With a short RNA (AUUU) or without RNA, AUH mainly exists as a trimer in solution. On the other hand, the AUH trimer dimerizes upon binding to one molecule of a long RNA containing 24 repeats of the AUUU motif, (AUUU)(24)A. AUH was crystallized with the long RNA. Although the RNA was disordered in the crystalline lattice, the AUH structure was determined as an asymmetric dimer of trimers with a kink in the alignment of the trimer axes, resulting in the formation of two clefts with significantly different sizes.     

The relative levels of translin-associated factor X (TRAX) and testis brain RNA-binding protein determine their nucleocytoplasmic distribution in male germ cells

Testis brain RNA-binding protein (TB-RBP), the mouse orthologue of human translin, is an RNA and single-stranded DNA-binding protein abundant in testis and brain. Translin-associated factor X (TRAX) was identified as a protein that interacts with TB-RBP and is dependent upon TB-RBP for stabilization. Using immunohistochemistry to investigate the subcellular locations of TB-RBP and TRAX during spermatogenesis, both proteins localize in nuclei in meiotic pachytene spermatocytes and in the cytoplasm of subsequent meiotic and post-meiotic cells. An identical subcellular distribution is seen in female germ cells. Western blot analysis of germ cell protein extracts reveals an increased ratio of TRAX to TB-RBP in meiotic pachytene spermatocytes compared with the post-meiotic round and elongated spermatids. Using COS-1 cells and mouse embryonic fibroblasts derived from TB-RBP null mice as model systems to examine the shuttling of TB-RBP and TRAX, we demonstrate that TRAX contains a functional nuclear localization signal and TB-RBP contains a functional nuclear export signal. Coexpression of both proteins in COS-1 cells and TB-RBP-deficient mouse embryonic fibroblasts reveals that the ratio of TRAX to TB-RBP determines their subcellular locations, i.e. increased TRAX to TB-RBP ratios lead to nuclear localizations, whereas TRAX remains in the cytoplasm when TB-RBP levels are elevated. These subcellular distributions require interaction between TB-RBP and TRAX. We propose that the subcellular locations of TB-RBP and TRAX in male germ cells are modulated by the relative ratios of TRAX and TB-RBP.     

Mutational analysis of the leucine zipper-like motif of the human immunodeficiency virus type 1 envelope transmembrane glycoprotein.

The N-terminal region of the envelope (env) transmembrane protein of human immunodeficiency virus type 1 (HIV-1) has a leucine zipper-like motif. This highly conserved zipper motif, which consists of a heptad repeat of leucine or isoleucine residues, has been suggested to play a role in HIV-1 env glycoprotein oligomerization. This hypothesis was tested by replacing the highly conserved leucine or isoleucine residues in the zipper motif with a strong alpha-helix breaker, proline. We report here that such substitutions did not abolish the ability of env protein to form oligomers, indicating that this highly conserved zipper motif does not have a crucial role in env protein oligomerization. However, the mutant viruses all showed impaired infectivity, suggesting that this conserved zipper motif can have an important role in the virus life cycle.     

A major determinant of hnRNP C protein binding to RNA is a novel bZIP-like RNA binding domain.

The C protein tetramer of hnRNP complexes binds approximately 150-230 nt of RNA with high cooperativity (McAfee J et al., 1996, Biochemistry 35:1212-1222). Three contiguously bound tetramers fold 700-nt lengths of RNA into a 19S triangular intermediate that nucleates 40S hnRNP assembly in vitro (Huang M et al., 1994, Mol Cell Biol 14:518-533). Although it has been assumed that the consensus RNA recognition motif (RRM) of C protein (residues 8-87) is the primary determinant of RNA binding, we report here that a recombinant construct containing residues 1-115 has very low affinity for RNA at physiological ionic strength (100 mM NaCl). Moreover, we demonstrate that an N-terminal deletion construct lacking the consensus RRM but containing residues 140-290 binds RNA with an affinity sufficient to account for the total free energy change observed for the binding of intact protein. Like native C protein, the 140-290 construct is a tetramer in solution and binds RNA stoichiometrically in a salt-resistant manner in 100-300 mM NaCl. Residues 140-179 of the N-terminal truncated variant contain 11 basic and 2 acidic residues, whereas residues 180-207 specify a leucine zipper motif that directs dimer assembly. Elements within the 50-residue carboxy terminus of C protein are required for tetramer assembly. A basic region followed by a leucine zipper is identical to the domain organization of the basic-leucine zipper (bZIP) class of DNA binding proteins. Sequence homologies with other proteins containing RRMs and the bZIP motif suggest that residues 140-207 represent a conserved bZIP-like RNA binding motif (designated bZLM). The steric orientation of four high-affinity RNA binding sites about rigid leucine zipper domains may explain in part C protein''s asymmetry, its large occluded site size, and its RNA folding activity.     

The DNA-binding domain of the Cys-3 regulatory protein of Neurospora crassa is bipartite.

Cys-3, the major sulfur regulatory gene of Neurospora crassa, encodes a regulatory protein that is capable of sequence-specific interaction with DNA. The interaction is mediated by a region within the CYS3 protein (the bzip region) which contains a potential dimer-forming surface, the leucine zipper, and an adjacent basic DNA contact region, NH2-terminal to the leucine zipper. To investigate the bipartite nature of the bzip region, a series of cys-3 mutants obtained by oligonucleotide-directed mutagenesis were expressed and tested for dimer formation as well as DNA binding and in vivo function. The results demonstrate that CYS3 protein exists as a dimer in the presence and absence of the target DNA and that dimerization of CYS3 is mediated strictly by the leucine zipper, which is required for both cys-3 function in vivo and DNA-binding activity in vitro. Furthermore, a truncated CYS3 protein corresponding to just the bzip region was found to mediate dimer formation and to possess DNA-binding activity. A CYS3 mutant protein with a pure methionine zipper showed significant, although reduced, function in vivo and in vitro.