Testis Brain RNA-binding Protein Research Articles

Testis-specific proteins, TSNAXIP1 and 1700010I14RIK, are important for sperm motility and male fertility in mice.

TSN (translin), also called testis brain RNA-binding protein, binds to TSNAX (translin-associated factor X) and is suggested to play diverse roles, such as RNA metabolism and DNA damage response. TSNAXIP1 (Translin-associated factor X-interacting protein 1) was identified as a TSNAX-interacting protein using a yeast two-hybrid system, but its function in vivo was unknown. To reveal the function of TSNAXIP1 in vivo in mice. We generated Tsnaxip1 knockout mice using the CRISPR/Cas9 system and analyzed their fertility and sperm motility. Further, we generated 1700010I14Rik knockout mice, because 1700010I14RIK is also predominantly expressed in testes and contains the same Pfam (protein families) domain as TSNAXIP1. Reduced male fertility and impaired sperm motility with asymmetric flagellar waveforms were observed in not only Tsnaxip1 but also 1700010I14Rik knockout mice. Unlike Tsn knockout mice, no abnormalities were found in testicular sections of either Tsnaxip1 or 1700010I14Rik knockout mice. Furthermore, TSNAXIP1 was detected in the sperm tail and fractionated with axonemal proteins. Unlike the TSN-TSNAX complex, whose disruption causes abnormal vacuoles in mouse testes, TSNAXIP1 and 1700010I14RIK may play roles in regulating sperm flagellar beating patterns.

Functional characterization of testis-brain RNA-binding protein, TB-RBP/Translin, in translational regulation

Testis-brain RNA-binding protein (TB-RBP/Translin) is known to contribute to the translational repression of a subset of haploid cell-specific mRNAs, including protamine 2 (Prm2) mRNA. Mutant mice lacking TB-RBP display abnormal spermatogenesis, despite normal male fertility. In this study, we carried out functional analysis of TB-RBP in mammalian cultured cells to understand the mechanism of translational repression by this RNA-binding protein. Although the amino acid sequence contained a eukaryotic translation initiation factor 4E (EIF4E)-recognition motif, TB-RBP failed to interact with EIF4E. In cultured cells, TB-RBP was unable to reduce the activity of luciferase encoded by a reporter mRNA carrying the 3’-untranslated region of Prm2. However, λΝ-BoxB tethering assay revealed that the complex of TB-RBP with its binding partner, Translin-associated factor X (TRAX), exhibits the ability to reduce the luciferase reporter activity by degrading the mRNA. These results suggest that TB-RBP may play a regulatory role in determining the sequence specificity of TRAX-catalyzed mRNA degradation.

The DNA/RNA-Binding Protein, Translin, Binds microRNA122a and Increases Its In Vivo Stability

Translin (TSN), also known as testis-brain RNA-binding protein, is proposed to bind to breakpoint junctions at chromosomal translocations in the nucleus and to specific RNAs in the cytoplasm. In germ cells of the mouse testis, it recognizes target mRNAs transcribed by the transcription factor CREM-tau in spermatids, specific meiotically expressed mRNAs, and a noncoding RNA that encodes piRNAs. Here we show that TSN also binds to the microRNA miR-122a. MiR-122a is expressed in late-stage germ cells and is complementary to a sequence in the 3' untranslated region of the transition protein 2 mRNA. The binding of TSN to miR-122a increases its in vivo stability, suggesting an additional posttranscriptional function for TSN.

Meiotic Messenger RNA and Noncoding RNA Targets of the RNA-Binding Protein Translin (TSN) in Mouse Testis1

In postmeiotic male germ cells, TSN, formerly known as testis brain-RNA binding protein, is found in the cytoplasm and functions as a posttranscriptional regulator of a group of genes transcribed by the transcription factor CREM-tau. In contrast, in pachytene spermatocytes, TSN is found predominantly in nuclei. Tsn-null males show a reduced sperm count and high levels of apoptosis in meiotic cells, suggesting a critical function for TSN during meiosis. To identify meiotic target RNAs that associate in vivo with TSN, we reversibly cross-linked TSN to RNA in testis extracts from 17-day-old and adult mice and immunoprecipitated the complexes with an affinity-purified TSN antibody. Extracts from Tsn-null mice were used as controls. Cloning and sequencing the immunoprecipitated RNAs, we identified four new TSN target mRNAs, encoding diazepam-binding inhibitor-like 5, arylsulfatase A, a tetratricopeptide repeat structure-containing protein, and ring finger protein 139. In contrast to the population of postmeiotic translationally delayed mRNAs that bind TSN, these four mRNAs are initially expressed in pachytene spermatocytes. In addition, anti-TSN also precipitated a nonprotein-coding RNA (ncRNA), which is abundant in nuclei of pachytene spermatocytes and has a putative polyadenylation signal, but no open reading frame. A second similar ncRNA is adjacent to a GGA repeat, a motif frequently associated with recombination hot spots. RNA gel-shift assays confirm that the four new target mRNAs and the ncRNA specifically bind to TSN in testis extracts. These studies have, for the first time, identified both mRNAs and a ncRNA as TSN targets expressed during meiosis.

Translin associated protein X is essential for cellular proliferation

DNA vectors that express short hairpin RNAs (shRNAs) provide a new tool for reverse genetic analysis for selective long-term reduction of gene expression in mammalian cells. Using shRNA constructs with a cytomegalovirus promoter and an actin intron between the hairpins for stabilization, we reduce expression of an exogenously expressed gene, GFP and the endogenous protein, Translin-associated factor X (TRAX), in stably transfected Hela cell lines. The reduction of TRAX in Hela cells causes reduced cell proliferation. This decrease is specific as there is no equivalent reduction of the TRAX interacting protein, Testis brain RNA-binding protein, or any significant increase in a number of interferon-related target genes.

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.

The kinesin KIF17b and RNA-binding protein TB-RBP transport specific cAMP-responsive element modulator-regulated mRNAs in male germ cells.

Testis brain RNA-binding protein (TB-RBP), the mouse orthologue of the human protein Translin, is a widely expressed and highly conserved protein with proposed functions in chromosomal translocations, mitotic cell division, and mRNA transport, stabilization, and storage. Targeted inactivation of TB-RBP leads to abnormalities in fertility and behavior. A testis-enriched kinesin KIF17b coimmunoprecipitates with TB-RBP in a RNA-protein complex containing specific cAMP-responsive element modulator (CREM)-regulated mRNAs. The specificity of this interaction is confirmed by in vivo RNA-protein crosslinking and transfections of hippocampal neurons. Combining in situ hybridization and immunohistochemistry at the electron microscope level, a temporally sequential dissociation of KIF17b and TB-RBP from specific mRNAs is detected with TB-RBP release coincident with the time of mRNA translation, indicating a separation of the processes of transport and translation. We conclude that KIF17b serves as a molecular motor component of a TB-RBP-mouse ribonucleoprotein complex transporting a group of specific CREM-regulated mRNAs in mammalian male postmeiotic germ cells. Because KIF17b has been reported to control CREM-dependent transcription in male germ cells by regulating the intracellular location of the transcriptional coactivator activator of CREM in testis, this indicates that one kinesin links the processes of transcription and transport of specific mRNAs in mammalian male germ cells.

Mice deficient for testis-brain RNA-binding protein exhibit a coordinate loss of TRAX, reduced fertility, altered gene expression in the brain, and behavioral changes.

Testis-brain RNA-binding protein (TB-RBP), the mouse orthologue of the human protein Translin, is a widely expressed and highly conserved protein with proposed functions in chromosomal translocations, mitotic cell division, and mRNA transport and storage. To better define the biological roles of TB-RBP, we generated mice lacking TB-RBP. Matings between heterozygotes gave rise to viable, apparently normal homozygous mutant mice at a normal Mendelian ratio. The TB-RBP-related and -interacting protein Translin-associated factor X was reduced to 50% normal levels in heterozygotes and was absent in TB-RBP-null animals. The null mice were 10 to 30% smaller than their wild-type or heterozygote littermates at birth and remained so to about 6 to 9 months of age, showed normal B- and T-cell development, and accumulated visceral fat. TB-RBP-null male mice were fertile and sired offspring but had abnormal seminiferous tubules and reduced sperm counts. Null female mice were subfertile and had reduced litter sizes. Microarray analysis of total brain RNA from null and wild-type mice revealed an altered gene expression profile with the up-regulation of 14 genes and the down-regulation of 217 genes out of 12,473 probe sets. Numerous neurotransmitter receptors and ion channels, including gamma-aminobutyric acid A receptor alpha1 and glutamate receptor alpha3, were strongly down-regulated. Behavioral abnormalities were also seen. Compared to littermates, the TB-RBP-null mice appeared docile and exhibited reduced Rota-Rod performance.

Mouse testis brain RNA-binding protein/translin selectively binds to the messenger RNA of the fibrous sheath protein glyceraldehyde 3-phosphate dehydrogenase-S and suppresses its translation in vitro.

The testis brain RNA-binding protein (TB-RBP/translin) is a DNA- and RNA-binding protein with multiple functions. As an RNA-binding protein, TB-RBP binds to conserved sequence elements often present in the 3' untranslated regions (UTRs) of specific mRNAs modulating their translation and transport. To identify additional mRNA targets of TB-RBP, immunoprecipitation and reverse transcription-polymerase chain reaction (RT-PCR) assays were carried out using an affinity-purified antibody to TB-RBP with testicular extracts. Gapds mRNA was found to be selectively precipitated in a TB-RBP-mRNA complex. Consistent with the delayed translation of GAPDS and the subcellular ribonucleoprotein location of TB-RBP, polysomal gradient analysis showed that most of the Gapds mRNA in adult testis extracts was present in the nonpolysomal fractions. In vitro translation assays revealed that Gapds mRNA translation was inhibited by recombinant TB-RBP or by a TB-RBP mutant protein, Nb, capable of binding RNA. No inhibition was seen with mutant forms of TB-RBP lacking domains required for RNA binding, including the TB-RBP Cb mutant and the C-terminal-truncated form of TB-RBP that disrupts the leucine zipper. As an additional indicator of the specificity of TB-RBP inhibition of Gapds mRNA translation, a putative TB-RBP binding H-element was deleted from the 5' UTR of the Gapds mRNA. No translational inhibition by recombinant TB-RBP was seen with Gapds mRNA lacking the H element. These data suggest that TB-RBP is involved in the posttranscriptional regulation of Gapds gene expression during spermiogenesis. Moreover, the Gapds mRNA is the first mRNA shown to have a functional TB-RBP binding site in its 5' UTR.

A TB-RBP and Ter ATPase Complex Accompanies Specific mRNAs from Nuclei through the Nuclear Pores and into Intercellular Bridges in Mouse Male Germ Cells

The testis brain RNA-binding protein (TB-RBP) functions as an RNA-binding protein in brain and testis, binding to conserved sequence elements present in specific mRNAs, such as protamine 1 and 2. We show here by RNA gel shift assays, immunoprecipitation, and by a novel in situ hybridization immunohistochemical technique that TB-RBP binds to AKAP4 mRNA in male mouse germ cells. AKAP4 is a component of the fibrous sheath and functions as a scaffolding protein in the sperm flagellum. AKAP4 is encoded by an X-linked gene, is expressed solely in postmeiotic (haploid) male germ cells, and is an essential protein in all spermatozoa, requiring its transport between spermatids as a protein or mRNA. AKAP4 mRNA forms a complex with TB-RBP and the Ter ATPase in nuclei and remains associated with these proteins as it exits nuclei into the cytoplasm and as it passes through intercellular bridges between spermatids. A similar mRNA-TB-RBP-Ter ATPase association is seen for protamine 2 mRNA, which is stored in the cytoplasm of postmeiotic germ cells about 7 days before translation. In contrast, no association is seen with PGK-2 mRNA which is initially transcribed early in meiosis with increased transcription in postmeiotic male germ cells. Although PGK-2 mRNA is subject to translational control, it lacks TB-RBP-binding sequences in its mRNA. The AKAP4 or protamine 2 mRNA–protein complexes dissociate in late-stage male germ cells when the mRNAs are translated. We propose that TB-RBP and the Ter ATPase are part of a complex that accompanies specific mRNAs in haploid mouse male germ cells in intracellular and intercellular movement. The temporal relationship of TB-RBP binding and mRNA inactivation in conjunction with the subsequent dissociation of the mRNA–protein complex at the time of mRNA translation suggests a role in translational suppression and/or mRNA stabilization.

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 Å crystal structure of mouse TB-RBP. The structure is predominantly α-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.

Mouse testis-brain RNA-binding protein (TB-RBP): expression, purification and crystal X-ray diffraction.

TB-RBP (testis-brain RNA-binding protein) is a mouse RNA-binding protein that controls the temporal and spatial expression of mRNA. Found most abundantly in brain and male germ cells in the testis, TB-RBP is known to suppress the translation of specific testicular and brain mRNAs as part of cell development. TB-RBP-mRNA complexes can bind microtubules and thereby facilitate RNA translocation. Translin is the human orthologue of TB-RBP which binds to single-stranded ends of DNA sequences in breakpoint regions of chromosomal translocations. TB-RBP/translin has been crystallized in space group P2(1)2(1)2. The expression, purification, and crystallization of TB-RBP are described as well as preliminary X-ray diffraction data. The multimeric state of TB-RBP is addressed using dynamic light-scattering results.

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.

Intracellular and intercellular transport of many germ cell mRNAs is mediated by the DNA- and RNA-binding protein, testis-brain-RNA-binding protein (TB-RBP).

Functions ranging from RNA transport and translational regulation to DNA rearrangement and repair have been proposed for the DNA- and RNA-binding protein, testis-brain-RNA-binding protein (TB-RBP). TB-RBP is primarily in the nuclei of male germ cells during meiosis and in the cytoplasm of male cells after metaphase I of meiosis. Based on its shift in subcellular locations as germ cells differentiate and its binding to microtubules and microfilaments, a model is presented proposing an involvement of TB-RBP in mRNA transport from nucleus to cytoplasm and in the sharing of mRNAs transcribed from the sex chromosomes by movement through intercellular bridges of germ cells.

Intracellular and intercellular transport of many germ cell mRNAs is mediated by the DNA‐ and RNA‐binding protein, testis‐brain‐RNA‐binding protein (TB‐RBP)

Intracellular and intercellular transport of many germ cell mRNAs is mediated by the DNA‐ and RNA‐binding protein, testis‐brain‐RNA‐binding protein (TB‐RBP)

Intracellular and intercellular transport of many germ cell mRNAs is mediated by the DNA‐ and RNA‐binding protein, testis‐brain‐RNA‐binding protein (TB‐RBP)

Intracellular and intercellular transport of many germ cell mRNAs is mediated by the DNA‐ and RNA‐binding protein, testis‐brain‐RNA‐binding protein (TB‐RBP)

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.

The suppression of testis-brain RNA binding protein and kinesin heavy chain disrupts mRNA sorting in dendrites

Ribonucleoprotein particles (RNPs) are thought to be key players in somato-dendritic sorting of mRNAs in CNS neurons and are implicated in activity-directed neuronal remodeling. Here, we use reporter constructs and gel mobility shift assays to show that the testis brain RNA-binding protein (TB-RBP) associates with mRNPs in a sequence (Y element) dependent manner. Using antisense oligonucleotides (anti-ODN), we demonstrate that blocking the TB-RBP Y element binding site disrupts and mis-localizes mRNPs containing (alpha)-calmodulin dependent kinase II (alpha)-CAMKII) and ligatin mRNAs. In addition, we show that suppression of kinesin heavy chain motor protein alters only the localization of (alpha)-CAMKII mRNA. Thus, differential sorting of mRNAs involves multiple mRNPs and selective motor proteins permitting localized mRNAs to utilize common mechanisms for shared steps.

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.

Testis–brain RNA-binding protein (Translin) is primarily expressed in neurons of the mouse brain

The subcellular location(s) of the DNA- and RNA-binding protein, Testis–Brain RNA-Binding Protein (TB-RBP)/Translin in mouse brain has been determined in paraffin sections by immunocytochemistry with an affinity purified antibody to mouse recombinant TB-RBP. Nuclear staining was frequently seen in neurons throughout the brain, but no TB-RBP/Translin was detected in many of the neurons in superficial layers of the cerebral cortex and in some cells of the cerebellum. Cytoplasmic staining extending into the dendrites was seen in large neurons such as pyramidal neurons in Layer 5 of the cortex and magnocellular neurons of the hypothalamus or the brainstem raphe.