cDNAs were cloned for the murine and human orthologues of Chlamydomonas PF20, a component of the alga axoneme central apparatus that is required for flagellar motility. The mammalian genes encode transcripts of 1.4 and 2.5 kb that are highly expressed in testis. The two transcripts appear to arise from alternative transcription start sites. The murine Pf20 gene was mapped to chromosome 1, syntenic with the location of the human gene on chromosome 2. An antibody generated against an N-terminal sequence of mouse Pf20 recognized a 71-kDa protein in sperm and testis extracts. Immunocytochemistry localized Pf20 to the tails of permeabilized sperm; electron microscope immunocytochemistry showed that Pf20 was located in the axoneme central apparatus. A murine Pf20-green fluorescent protein fusion protein expressed in Chinese hamster ovary cells accumulated in the cytoplasm. When coexpressed with Spag6, the mammalian orthologue of Chlamydomonas PF16, Pf20 was colocalized with Spag6 on polymerized microtubules. Yeast two-hybrid assays demonstrated interaction of the Pf20 WD repeats with Spag6. Pf20 was markedly reduced in sperm collected from mice lacking Spag6, which are infertile due to a motility defect. Our observations provide the first evidence for an association between mammalian orthologues of two Chlamydomonas proteins known to be critical for axoneme structure and function.The "9 ϩ 2" microtubule architecture of the eukaryote axoneme has remained virtually unchanged over millions of years of evolution. Understanding the function of molecules that make up the axoneme is important for elucidating the assembly and activity of these structures that are essential for cell motility. The distinctive arrangement of nine outer doublet microtubules in a circle around a central pair of microtubules is recognizable in electron micrographs of flagella and cilia from plants, algae, protists, and animals. Attached along specific microtubules at precise locations and intervals are ranks of substructures including dynein arms, radial spokes, and central pair projections (25,26). Axonemal dyneins form the inner and outer arm structures that have different functions; the outer arms add power and adjust beat frequency (3,4,10,15,16,24,33); the inner arms generate the axonemal waveform (4,7,14,17,27). To work together efficiently, the multiple dynein isoforms must be locally activated and inactivated at different points in the beat cycle, both around the axoneme and along its length.Structural and genetic evidence implicated the radial spokecentral pair structures as key regulators of dynein activity. The radial spoke heads make transient contact with structures that project from the central pair microtubules (35). The central pair is composed of two microtubules (designated C1 and C2 in algae) and their associated structures which include the central pair projections, central pair bridges linking the two tubules, and central pair caps which are attached to the distal or plus ends of the microtubules.Mutants of the alga Chlamydomon...

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 ␥-aminobutyric acid A receptor ␣1 and glutamate receptor ␣3, 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.The mouse nucleic acid-binding protein testis-brain RNAbinding protein (TB-RBP) is the orthologue of the human protein Translin (5, 6, 26). The official nomenclature for the mouse TB-RBP gene is Tsn. Translin/TB-RBP is expressed in many organisms, including fission yeasts, plants, frogs, insects, and mammals. In mammalian tissues, it is ubiquitously expressed, with especially high levels in the brain and testis (18,31,32). Translin is a 228-amino-acid protein encoded by a single-copy gene on human chromosome 2 (3). TB-RBP, encoded by a single-copy gene on mouse chromosome 1, differs from the human protein in three amino acids (3,55,56). Translin has been implicated in DNA rearrangements through binding to single-stranded DNA sequences found at the breakpoint junctions of chromosomal translocations in lymphoid malignancies and solid tumors (26). Electron microscopic and analytical ultracentrifugation studies have revealed multimeric ring structures of Translin which have been proposed to recognize staggered breaks occurring at recombination hot spots in the genome (26,35,45,52). Similar multimeric ring structures are seen when TB-RBP is crystallized (44). Recently, Translin was shown to accelerate cell proliferation when overexpressed in cultured HEK cells (22).In addition to its proposed roles in DNA recombination and cell proliferation, TB-RBP functions in mRNA transport and/or stabilization and in translational regulation (40,41). In the brain and testis, TB-RBP serves as a linker protein binding specific mRNAs t...

Uterine fibroids, also known as uterine leiomyoma (UL), are monoclonal tumors of the smooth muscle tissue layer (myometrium) of the uterus. Although ULs are considered benign, uterine fibroids are the source of major quality-of-life issues for approximately 25% of all women, who suffer from clinically significant symptoms of UL. Despite the prevalence of UL, there is no treatment option for UL which is long term, cost-effective, and leaves fertility intact. The lack of understanding about the etiology of UL contributes to the scarcity of medical therapies available. Studies have identified an important role for sex steroid hormones in the pathogenesis of UL, and have driven the use of hormonal treatment for fibroids, with mixed results. Dysregulation of cell signaling pathways, miRNA expression, and cytogenetic abnormalities have also been implicated in UL etiology. Recent discoveries on the etiology of UL and the development of relevant genetically modified rodent models of UL have started to revitalize UL research. This review outlines the major characteristics of fibroids; major contributors to UL etiology, including steroid hormones; and available preclinical animal models for UL.

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-TB⅐RBP interactions modulate the DNA-and RNA-binding activity of TB-RBP.The process of mammalian spermatogenesis is highly organized spatially and temporally. Highly controlled transcription and protein expression occur in each developmental stage. During the haploid interval, spermiogenesis, the spermatids become transcriptionally inactive, although there is a need for the synthesis of many proteins essential for the formation of spermatozoa (1). The sex chromosomes encode numerous genes essential for gametogenesis. Because the spermatids are haploid cells, they contain either the X or Y chromosome. Thus, intercellular transport of mRNA in the haploid cells is a critical process to ensure genetic equivalency.The testis brain RNA-binding protein (TB-RBP) 1 was identified and cloned on the basis of its ability to bind H and Y sequence elements in the 3Ј-untranslated repeats of mouse protamine 1 and 2 mRNAs (2). TB-RBP is the mouse orthologue of human Translin, a single-stranded DNA-binding protein that binds consensus sequence breakpoint junctions of chromosomal translocations in lymphoid malignancies (3). The TB-RBP/Translin consensus binding sequences are also found in TLS-CHOP reciprocal translocations, in therapy-related translocations in acute myeloid leukemias, and in BCR-ABL translocations in chronic myeloid leukemia (4 -6). Sequence analysis in a meiotic recombination hot spot region of human chromosome 16 shows TB-RBP/Translin binding sequences near the breakpoint (7). TB-RBP/Translin has also been proposed to act as a single-stranded DNA-binding transcription factor, which activates early response gene expression in the brain (8). TB-RBP/Translin also functions as a RNA-binding protein mediating intracellular and intercellular mRNA transport (9, 10). RNA binding of TB-RBP has been observed in brain and testis, and the binding is dependent upon Y and H sequence elements (2). Many testis-and brain-specific mRNAs have Y and H consensus sequences, and specific RNA⅐TB-RBP inter...

Uterine fibroids (leiomyomas) are the most common tumors of the female reproductive tract, occurring in up to 77% of reproductiveaged women, yet molecular pathogenesis remains poorly understood. A role for atypically activated mammalian target of rapamycin (mTOR) pathway in the pathogenesis of uterine fibroids has been suggested in several studies. We identified that G protein-coupled receptor 10 [GPR10, a putative signaling protein upstream of the phosphoinositide 3-kinase-protein kinase B/AKT-mammalian target of rapamycin (PI3K/AKT-mTOR) pathway] is aberrantly expressed in uterine fibroids. The activation of GPR10 by its cognate ligand, prolactin releasing peptide, promotes PI3K-AKT-mTOR pathways and cell proliferation specifically in cultured primary leiomyoma cells. Additionally, we report that RE1 suppressing transcription factor/neuron-restrictive silencing factor (REST/NRSF), a known tumor suppressor, transcriptionally represses GPR10 in the normal myometrium, and that the loss of REST in fibroids permits GPR10 expression. Importantly, mice overexpressing human GPR10 in the myometrium develop myometrial hyperplasia with excessive extracellular matrix deposition, a hallmark of uterine fibroids. We demonstrate previously unrecognized roles for GPR10 and its upstream regulator REST in the pathogenesis of uterine fibroids. Importantly, we report a unique genetically modified mouse model for a gene that is misexpressed in uterine fibroids.

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.

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. D uring spermatogenesis, male germ cells undergo massive differentiation and rapid polarization that require transport and specific localization of vesicles, proteins, mRNAs, and organelles. The need to maintain phenotypic equivalence of the X or Y chromosome bearing haploid male germ cells further necessitates the sharing of specific mRNAs through intercellular bridges (1, 2). A number of kinesin and dynein superfamily motor proteins have been proposed to carry out transport processes in the testis (3). The mRNA binding protein TB-RBP, the mouse orthologue of human Translin (4), binds specific mRNAs in postmeiotic germ cells and in neurons, two of the highly polarized cell types in which mRNA transport and localization are essential. In male germ cells TB-RBP transports specific mRNAs intracellularly and intercellularly in a mouse ribonucleoprotein complex containing the Ter ATPase (5). Microtubule reconstitution experiments reveal that specific translationally delayed mRNAs are linked to microtubules through TB-RBP (6). TB-RBP binds brain mRNAs such as ␣-Ca 2ϩ -calmodulin-dependent protein kinase II mRNA and ligatin (7,8) and a number of translationally delayed and X chromosome-encoded mRNAs that are solely expressed in male postmeiotic germ cells (5). In our investigation of the components of the TB-RBP-mRNA complexes involved in mRNA transport, we have identified a kinesin by coimmunoprecipitation and matrix-assisted laser desorption͞ionization-time-offlight (MALDI-TOF) mass spectroscopy. This kinesin, KIF17b, is an isoform of the neuronal...

slow cell growth rates. We conclude that TRAX is posttranscriptionally stabilized by TB-RBP and both proteins are needed for normal cell proliferation.The human protein Translin and its mouse orthologue, testis-brain RNA-binding protein (TB-RBP), 1 are single-stranded DNA-and RNA-binding proteins with proposed functions in chromosomal translocations in lymphoid cells and mRNA transport and storage in brain and testis (1-4). TB-RBP is a highly conserved protein with mouse and human proteins differing in 3 of 228 amino acids (5, 6). In in vitro assays, TB-RBP/Translin binds to consensus chromosomal DNA breakpoint junctions as an octameric ring and recognizes DNA breaks at genomic hotspots (2,7,8). TB-RBP also binds to consensus RNA sequences present in many brain and testis mRNAs (6, 9 -11) and links specific mRNAs to microtubules (10,12,13). In the testis TB-RBP forms a complex with the Ter ATPase to transport specific mRNAs from nuclei to cytoplasm and through intercellular bridges (4). This ribonucleoprotein complex contains the kinesin KIF 17b, a testicular motor protein controlling transcription of CREM-regulated male germ cell mRNAs (14). The level of Translin has been demonstrated to closely parallel the proliferative state of somatic cells with its induced overexpression in HEK 293 cells accelerating cell proliferation (15). TRAX, first characterized as a Translin-like protein (16), forms DNA and RNA binding complexes with Translin (17,18). TRAX has been proposed to function in DNA repair in conjunction with the nuclear matrix protein C1D (19).Recently, we generated mice with a functional deletion of the TB-RBP gene (20). The mice are growth retarded and show defects in behavior and fertility. Many of the germ cells in the testis cannot proceed beyond first meiotic metaphase suggesting a defect in chromosome segregation and cytokinesis. Using 14.5-day-old embryos from heterozygous crosses, we have developed lines of MEFs from TB-RBP null animals and from their littermates. As in TB-RBP-deficient mice, these cells lack both TB-RBP and TRAX, although TRAX mRNA levels are not reduced. We find that early passage null MEFs have slower rates of proliferation with a block in the G 2 stage of the cell cycle. Reintroduction of TB-RBP increases the rate of cell proliferation and stabilizes the TRAX protein. Transgenic and transfection experiments indicate that TRAX protein is dependent upon TB-RBP for stabilization and both TB-RBP and TRAX are needed for normal cell proliferation. EXPERIMENTAL PROCEDURES Generation of MEFs-TB-RBP-deficientMEFs were derived from TB-RBP null mice generated by gene trapping (20). MEFs were produced using established protocols (21). Briefly, embryos were removed from pregnant heterozygous mice at 14.5 days and rinsed separately in sterile PBS. Fetal membranes, placenta, head, and soft tissues were discarded, and the remaining tissues were rinsed in fresh PBS. Each embryo was minced and incubated at 37°C for 5 min in 2 ml of 0.25% trypsin/EDTA. Debris was removed by passing the cell susp...