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.     

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.     

Trax is a component of the Translin-containing RNA binding complex

Translin is a nucleic acid binding protein that has been implicated in regulating the targeting and translation of dendritic RNA. In previous studies, we found that Translin and its partner protein, Trax, are components of a gel-shift complex that is highly enriched in brain extracts. In those studies, we employed a DNA oligonucleotide, GS1, as a probe to label the complex. Translin has also been identified as a component of a gel-shift complex detected using an RNA oligonucleotide probe, derived from the 3' UTR of protamine-2 mRNA. Although we had assumed that these probes labeled the same complex, recent studies indicate that association of Trax with Translin suppresses its RNA binding activity. As these findings challenge this assumption and suggest that the native RNA binding complex does not contain Trax, we have re-examined this issue. We have found that the gel-shift complexes labeled with either GS1 or protamine-2 probes are "supershifted" by addition of Trax antibodies, indicating that both are heteromeric Translin/Trax complexes. In addition, cross-competition studies provide additional evidence that these probes label the same complex. Furthermore, analysis of recombinant Translin/Trax complexes generated by co-transfection of Trax with Translin in hEK293T demonstrates that they are labeled with either probe. Although recombinant Translin forms a homomeric nucleic acid binding complex in vitro, our findings indicate that both Trax and Translin are components of the native gel-shift complex labeled with either GS1 or protamine-2 probes.     

The Protein-Binding Potential of C2H2 Zinc Finger Domains

There are over 10,000 C2H2-type zinc finger (ZF) domains distributed among more than 1,000 ZF proteins in the human genome. These domains are frequently observed to be involved in sequence-specific DNA binding, and uncharacterized domains are typically assumed to facilitate DNA interactions. However, some ZFs also facilitate binding to proteins or RNA. Over 100 Cys2-His2 (C2H2) ZF-protein interactions have been described. We initially attempted a bioinformatics analysis to identify sequence features that would predict a DNA- or protein-binding function. These efforts were complicated by several issues, including uncertainties about the full functional capabilities of the ZFs. We therefore applied an unbiased approach to directly examine the potential for ZFs to facilitate DNA or protein interactions. The human OLF-1/EBF associated zinc finger (OAZ) protein was used as a model. The human O/E-1-associated zinc finger protein (hOAZ) contains 30 ZFs in 6 clusters, some of which have been previously indicated in DNA or protein interactions. DNA binding was assessed using a target site selection (CAST) assay, and protein binding was assessed using a yeast two-hybrid assay. We observed that clusters known to bind DNA could facilitate specific protein interactions, but clusters known to bind protein did not facilitate specific DNA interactions. Our primary conclusion is that DNA binding is a more restricted function of ZFs, and that their potential for mediating protein interactions is likely greater. These results suggest that the role of C2H2 ZF domains in protein interactions has probably been underestimated. The implication of these findings for the prediction of ZF function is discussed.     

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.     

RRM proteins interacting with the cap region of topoisomerase I

RNA recognition motif (RRM) domains bind both nucleic acids and proteins. Several proteins that contain two closely spaced RRM domains were previously found in protein complexes formed by the cap region of human topoisomerase I, a nuclear enzyme responsible for DNA relaxation or phosphorylation of SR splicing proteins. To obtain molecular insight into specific interactions between the RRM proteins and the cap region of topo I we examined their binary interactions using the yeast two-hybrid system. The interactions were established for hnRNP A1, p54(nrb) and SF2/ASF, but not for hnRNP L or HuR. To identify the amino acid pattern responsible for binding, experimental mutagenesis was employed and computational modelling of these processes was carried out. These studies revealed that two RRM domains and six residues of the consensus sequence are required for the binding to the cap region. On the basis of the above data, a structural model for the hnRNP A1-topoisomerase I complex was proposed. The main component of the hnRNP A1 binding site is a hydrophobic pocket on the beta-surface of the first RRM domain, similar to that described for Y14 protein interacting with Mago. We demonstrated that the interaction between RRM domains and the cap region was important for the kinase reaction catalyzed by topoisomerase I. Together with the previously described inhibitory effect of RRM domains of SF2/ASF on DNA cleavage, the above suggests that the binding of RRM proteins could regulate the activity of topoisomerase I.     

Somatodendritic localization of Translin, a component of the Translin/Trax RNA binding complex

Recent studies implicating dendritic protein synthesis in synaptic plasticity have focused attention on identifying components of the molecular machinery involved in processing dendritic RNA. Although Translin was originally identified as a protein capable of binding single-stranded DNA, subsequent studies have demonstrated that it also binds RNA in vitro. Because previous studies indicated that Translin-containing RNA/single-stranded DNA binding complexes are highly enriched in brain, we and others have proposed that it may be involved in dendritic RNA processing. To assess this possibility, we have conducted studies aimed at defining the localization of Translin and its partner protein, Trax, in brain. In situ hybridization studies demonstrated that both Translin and Trax are expressed in neurons with prominent staining apparent in cerebellar Purkinje cells and neuronal layers of the hippocampus. Subcellular fractionation studies demonstrated that both Translin and Trax are highly enriched in the cytoplasmic fraction compared with nuclear extracts. Furthermore, immunohistochemical studies with Translin antibodies revealed prominent staining in Purkinje neuron cell bodies that extends into proximal and distal dendrites. A similar pattern of somatodendritic localization was observed in hippocampal and neocortical pyramidal neurons. These findings demonstrate that Translin is expressed in neuronal dendrites and therefore support the hypothesis that the Translin/Trax complex may be involved in dendritic RNA processing.     

Co-localization of Trax and Mea2 in Golgi complex of pachytene spermatocytes in the mouse.

A golgin family protein, Mea2, is expressed at enhanced level in pachytene spermatocytes and is indispensable for mouse spermatogenesis. Because Trax was shown to interact with Mea2 in yeast two-hybrid, we investigated the localization of Trax in pachytene spermatocytes with immunofluorescent staining. Trax was found to accumulate in the Golgi complex of mid-late pachytene spermatocytes and intermingled with granular Mea2 signal in the central region. In a subline of the Mea2 mutant mouse, a truncated form of Mea2 devoid of the N-terminal region, DeltaMea2, was expressed. It localized to the rim of Golgi complex and thus occupied a region separate from that of Trax.     

KIF2Abeta: A kinesin family member enriched in mouse male germ cells, interacts with translin associated factor-X (TRAX)

Translin associated factor X (TRAX) is a binding partner of TB-RBP/Translin. A cDNA encoding the 260 C-terminal amino acids of KIF2Abeta was isolated from mouse testis cDNAs in a yeast two-hybrid library screen for specific TRAX-interacting proteins. KIF2Abeta was expressed predominantly in the mouse testis and enriched in germ cells. The interaction of full-length KIF2Abeta or its C-terminus with TRAX was verified using in vitro synthesized fusion proteins. Deletion mapping of the TRAX-binding region of KIF2Abeta indicated that amino acids 514-659 were necessary and sufficient for the interaction in vivo. Confocal microscopy studies using GFP-fusion proteins demonstrated that KIF2Abeta colocalizes with TRAX in a perinuclear location. KIF2Abeta does not interact with TB-RBP, suggesting that either TRAX can function as an adaptor molecule for motor proteins and TB-RBP, or that this interaction reveals an undescribed role for TRAX in germ cells. The interaction with KIF2Abeta suggests a role for TRAX in microtubule-based functions during spermatogenesis.     

The polymerization mechanism of the bacterial cell division protein FtsZ

Translin and Trax are components of an RNA binding complex initially detected in extracts of brain and testes. Although other tissues appear to contain much lower or negligible levels of the Translin/Trax gel-shift complex, we found, unexpectedly, that several of these peripheral tissues express Translin and Trax proteins at levels comparable to those present in brain. In this study, we demonstrate that the paradoxically low levels of the Translin/Trax complex in kidney and other peripheral tissues are due to masking of these sites by endogenous RNA. Thus, these findings indicate that the Translin/Trax complex is involved in RNA processing in a broader range of tissues than previously recognized.     

The phox homology (PX) domain protein interaction network in yeast

The phox homology (PX) domain is a phosphoinositide-binding domain that is conserved from yeast to human. Here we show for the first time by genome-wide two-hybrid screens and in vitro binding assays that the PX domain is a bona fide protein interaction domain. The yeast PX domain-only proteins Grd19p (YOR357C) and Ypt35p (YHR105W), as well as the isolated PX domains from Mvp1p (YMR004W), Snx42p/Cvt20p/Atg20p (YDL113C), Vam7p (YGL212W), and Vps17p (YOR132W), yielded a total of 40 reproducible two-hybrid interactions. Thirty-five interactions were found for the full-length proteins of Bem1p (YBR200W), Snx42p, Snx4p/Cvt13p (YJL036W), Vam7p, Vps5p (YOR069W), and Vps17p, but these appear not to require the PX domain, because these interactions could not be reproduced with PX-only baits. Interactions of Grd19p, Vam7p, Vps5p, Vps17p, and Ypt35p with members of the Yip1p family of proteins were detected consistently and were verified by in vitro binding assays. The N-terminal cytoplasmic domain of Yip1p and Yif1p mediates these interactions with PX domains. A mutation in the lipid-binding pocket of Ypt35p that reduces lipid binding markedly does not affect these PX domain protein interactions, arguing that lipid binding uses a different interaction surface than protein binding.     

Basic residues of the retroviral nucleocapsid play different roles in gag-gag and Gag-Psi RNA interactions

The Orthoretrovirus Gag interaction (I) domain maps to the nucleocapsid (NC) domain in the Gag polyprotein. We used the yeast two-hybrid system to analyze the role of Alpharetrovirus NC in Gag-Gag interactions and also examined the efficiency of viral assembly and release in vivo. We could delete either or both of the two Cys-His (CH) boxes without abrogating Gag-Gag interactions. We found that as few as eight clustered basic residues, attached to the C terminus of the spacer peptide separating the capsid (CA) and NC domains in the absence of NC, was sufficient for Gag-Gag interactions. Our results support the idea that a sufficient number of basic residues, rather than the CH boxes, play the important role in Gag multimerization. We also examined the requirement for basic residues in Gag for packaging of specific packaging signal (Psi)-containing RNA. Using a yeast three-hybrid RNA-protein interaction assay, second-site suppressors of a packaging-defective Gag mutant were isolated, which restored Psi RNA binding. These suppressors mapped to the p10 or CA domains in Gag and resulted in either introduction of a positively charged residue or elimination of a negatively charged one. These results imply that the structural interactions of NC with other domains of Gag are necessary for Psi RNA binding. Taken together, our results show that while Gag assembly only requires a certain number of positively charged amino acids, Gag binding to genomic RNA for packaging requires more complex interactions inherent in the protein tertiary structure.     

Interactions in the error-prone postreplication repair proteins hREV1, hREV3, and hREV7

Most mutations after DNA damage in yeast Saccharomyces cerevisiae are induced by error-prone translesion DNA synthesis employing scRev1 and DNA polymerase zeta that consists of scRev3 and scRev7 proteins. Recently, the human REV1 (hREV1) and REV3 (hREV3) genes were identified, and their products were revealed to be involved in UV-induced mutagenesis, as observed for their yeast counterparts. Human REV7 (hREV7) was also cloned, and its product was found to interact with hREV3, but the biological function of hREV7 remained unknown. We report here the analyses of precise interactions in the human REV proteins. The interaction between hREV1 and hREV7 was identified by the yeast two-hybrid library screening using a bait of hREV7, which was confirmed by in vitro and in vivo binding assays. The homodimerization of hREV7 was also detected in the two-hybrid analysis. In addition, the precise domains for interaction between hREV7 and hREV1 or hREV3 and for hREV7 homodimerization were determined. Although hREV7 interacts with both hREV1 and hREV3, a stable complex formation of the three proteins was undetectable in vitro. These findings suggest the possibility that hREV7 might play an important role in regulating the enzymatic activities of hREV1 and hREV3 for mutagenesis in response to DNA damage.     

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.     

Two dimerization domains in the trans-activation response RNA-binding protein (TRBP) individually reverse the protein kinase R inhibition of HIV-1 long terminal repeat expression.

Trans-activation response (TAR) RNA-binding protein (TRBP) is a cellular protein that binds to the human immunodeficiency virus-1 (HIV-1) TAR element RNA. It has two double-stranded RNA binding domains (dsRBDs), but only one is functional for TAR binding. TRBP interacts with the interferon-induced protein kinase R (PKR) and inhibits its activity. We used the yeast two-hybrid assay to map the interaction sites between the two proteins. We show that TRBP and PKR-N (178 first amino acids of PKR) interact with PKR wild type and inhibit the PKR-induced yeast growth defect in this assay. We characterized two independent PKR-binding sites in TRBP. These sites are located in each dsRBD in TRBP, indicating that PKR-TRBP interaction does not require the RNA binding activity present only in dsRBD2. TRBP and its fragments that interact with PKR reverse the PKR-induced suppression of HIV-1 long terminal repeat expression. In addition, TRBP activates the HIV-1 long terminal repeat expression to a larger extent than the addition of each domain. These data suggest that TRBP activates gene expression in PKR-dependent and PKR-independent manners.