ABSTRACT
Here we use an in vivo cross-linking and immunoprecipitation procedure to detect RNA targets of the multifunctional RNA-binding protein polypyrimidine tract-binding protein (PTBP) 2 in mouse testis. Eleven known mRNAs, including Ptbp2 mRNA, 28 RNAs matching intron sequences, and 12 small RNAs and repeat sequences are identified. The specificity of interaction between PTBP2 and its target RNAs was confirmed using RNA interference with mouse N2A cells. Reduction of PTBP2 levels led to decreases in 7 of 10 of the mRNAs, to the repression of alternative splicing of introns, and to reductions in specific miRNAs.
Subject(s)
MicroRNAs/metabolism , Nerve Tissue Proteins/metabolism , Polypyrimidine Tract-Binding Protein/metabolism , RNA, Messenger/metabolism , Testis/metabolism , Alternative Splicing/physiology , Animals , Base Sequence , Cell Line , Cross-Linking Reagents/pharmacology , Gene Expression Regulation , Immunoprecipitation/methods , Inteins , Male , Mice , Molecular Sequence Data , Protein Binding , Substrate SpecificityABSTRACT
The germ cell-specific DNA/RNA-binding protein MSY2 binds small RNAs (MSY-RNAs) that are approximately 25-31 nt in length, often initiate with a 5' adenine, and are expressed in both germ cells and somatic cells. MSY-RNA levels do not decrease in Miwi or Msy2 null mice. Most MSY-RNAs map within annotated genes, but some are PIWI-interacting RNA (piRNA)-like and map to piRNA clusters. MSY-RNAs are in both nuclei and cytoplasm. In nuclei, MSY-RNAs are enriched in chromatin, and in the cytoplasm they are detected in both ribonucleoproteins and polysomes.
Subject(s)
Proteins/metabolism , RNA-Binding Proteins/metabolism , RNA/metabolism , Testis/metabolism , Animals , Argonaute Proteins , Base Sequence , Cell Nucleus/metabolism , Chromatin/metabolism , Chromosome Mapping , Cytoplasm/metabolism , Electrophoresis, Polyacrylamide Gel , Gene Expression Regulation, Developmental , Genomics/methods , Immunoprecipitation , Male , Mice , Mice, Knockout , Polyribosomes/metabolism , Protein Binding , Proteins/genetics , RNA/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Reverse Transcriptase Polymerase Chain Reaction , Ribonucleoproteins/metabolism , Testis/cytology , Testis/growth & developmentABSTRACT
Phosphoglycerate kinase 2 (PGK2) is a germ cell-specific protein whose mRNA is translationally regulated in the mammalian testis. Using RNA affinity chromatography with the 3'-untranslated region (UTR) of Pgk2 mRNA and adult testis extracts, several associated proteins including a novel isoform of the AU-rich element RNA-binding protein and KH-type splicing regulatory protein (KSRP) were identified. KSRP, a protein of approximately 75 kDa, is widely expressed in somatic and germ cells where it is primarily nuclear. In addition to the approximately 75-kDa KSRP, a approximately 52-kD KSRP, t-KSRP, is present in the cytoplasm of a subpopulation of germ cells. t-KSRP binds directly to a 93-nt sequence (designated the F1 region) of the 3'-UTR of the Pgk2 mRNA and destabilizes Pgk2 mRNA constructs in testis extracts and in transfected cells. We conclude that this testicular variant of the multifunctional nucleic acid-binding protein, KSRP, serves as a decay-promoting factor for Pgk2 mRNA in male germ cells.
Subject(s)
Isoenzymes/genetics , Phosphoglycerate Kinase/genetics , RNA Stability , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Testis/metabolism , Trans-Activators/metabolism , 3' Untranslated Regions/chemistry , 3' Untranslated Regions/metabolism , Animals , Binding Sites , Cytoplasm/metabolism , Genetic Variation , HeLa Cells , Humans , Isoenzymes/metabolism , Male , Meiosis , Mice , Nerve Tissue Proteins/metabolism , Phosphoglycerate Kinase/metabolism , Polypyrimidine Tract-Binding Protein/metabolism , Protein Isoforms/genetics , Protein Isoforms/metabolism , RNA-Binding Proteins/isolation & purification , Testis/enzymology , Trans-Activators/isolation & purificationABSTRACT
Degradation of maternal mRNA is thought to be essential to undergo the maternal-to-embryonic transition. Messenger RNA is extremely stable during oocyte growth in mouse and MSY2, an abundant germ cell-specific RNA-binding protein, likely serves as a mediator of global mRNA stability. Oocyte maturation, however, triggers an abrupt transition in which most mRNAs are significantly degraded. We report that CDC2A (CDK1)-mediated phosphorylation of MSY2 triggers this transition. Injecting Cdc2a mRNA, which activates CDC2A, overcomes milrinone-mediated inhibition of oocyte maturation, induces MSY2 phosphorylation and the maturation-associated degradation of mRNAs. Inhibiting CDC2A following its activation with roscovitine inhibits MSY2 phosphorylation and prevents mRNA degradation. Expressing non-phosphorylatable dominant-negative forms of MSY2 inhibits the maturation-associated decrease in mRNAs, whereas expressing constitutively active forms induces mRNA degradation in the absence of maturation and phosphorylation of endogenous MSY2. A positive-feedback loop of CDK1-mediated phosphorylation of MSY2 that leads to degradation of Msy2 mRNA that in turn leads to a decrease in MSY2 protein may ensure that the transition is irreversible.
Subject(s)
CDC2 Protein Kinase/metabolism , Oocytes/metabolism , RNA Stability , RNA, Messenger, Stored/metabolism , RNA-Binding Proteins/metabolism , Animals , Mice , Phosphorylation , RNA-Binding Proteins/genetics , Ribonucleases/metabolismABSTRACT
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.
Subject(s)
DNA-Binding Proteins/metabolism , MicroRNAs/metabolism , RNA Stability , RNA-Binding Proteins/metabolism , Animals , Cell Line , Gene Expression Regulation , Male , Mice , Protein Binding , Testis/metabolismABSTRACT
MSY2 is a highly conserved and abundant DNA/RNA-binding protein that functions as a global stabilizer/translational suppressor of mRNAs in male germ cells. The polypyrimidine tract binding protein, PTBP2, is an RNA-binding protein that splices nuclear transcripts and stabilizes specific mRNAs in the cytoplasm. The mechanisms whereby MSY2 selects and stabilizes a large group of male germ cell mRNAs and PTBP2 stabilizes specific mRNAs such as the phosphoglycerate kinase 2 mRNA in the testis and in transfected cells will be discussed.
Subject(s)
Nerve Tissue Proteins/physiology , Polypyrimidine Tract-Binding Protein/physiology , RNA, Messenger/genetics , RNA-Binding Proteins/physiology , Testis/metabolism , Animals , Humans , Isoenzymes/genetics , Male , Phosphoglycerate Kinase/geneticsABSTRACT
The mammalian testis expresses a class of small noncoding RNAs that interact with mammalian PIWI proteins. In mice, the PIWI-interacting RNAs (piRNAs) partner with mammalian PIWI proteins, PIWIL1 and PIWIL2, also known as MIWI and MILI, to maintain transposon silencing in the germline genome. Here, we demonstrate that inactivation of Nct1/2, two noncoding RNAs encoding piRNAs, leads to derepression of LINE-1 (L1) but does not affect mouse viability, spermatogenesis, testicular gene expression, or fertility. These findings indicate that piRNAs from a cluster on chromosome 2 are necessary to maintain transposon silencing.
Subject(s)
DNA Transposable Elements/genetics , Long Interspersed Nucleotide Elements/genetics , RNA, Small Interfering/genetics , RNA, Small Interfering/physiology , Animals , Gene Expression Profiling , Gene Expression Regulation/genetics , Gene Silencing , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Multigene Family/physiology , Mutation/physiology , Oligonucleotide Array Sequence Analysis , Reproduction/genetics , Testis/metabolismABSTRACT
In eukaryotic cells, the vast majority of transcribed sequences are extragenic with no known functions. Translin is a DNA/RNA-binding protein involved in mRNA transport and translation in postmeiotic male germ cells. In an effort to identify meiotic target RNAs of Translin, reversible RNA protein cross-linking and immunoprecipitations with an affinity purified antibody to Translin were performed. Four new meiotically expressed mRNAs and one noncoding RNA with Translin binding sites were identified. Following sequencing, the noncoding RNA, Nct1, was 100% identical to a site on mouse chromosome 2. A second partially homologous sequence, Nct2, was detected nearby. Nct 1 and 2 contained sequences identical to piRNAs. Nct1 and 2 appear to be male germ cell-specific transcripts and are predominantly detected in pachytene spermatocytes. Focusing on the abundant single-copy PIWI-interacting RNA (piRNA), germline small RNA (gsRNA10) (the gsRNA10 sequence is identical to 29 nt in Nct1), we find that gsRNA10 increases greatly as spermatogenesis proceeds with concomitant decreases in Nct1 and 2. The piRNA gsRNA10 binds to the germ cell-specific Y-box protein, MSY2, but not to Translin. Although the size of the primary transcript(s) encoding the piRNAs in the locus on chromosome 2 is not known, we propose that Nct1 and 2 are part of a piRNA precursor.
Subject(s)
Meiosis/genetics , RNA, Small Interfering/genetics , RNA, Untranslated/metabolism , Testis/metabolism , Animals , Humans , Male , Mammals/genetics , Mice , Mice, Inbred Strains , Protein Binding , RNA, Small Interfering/metabolism , RNA, Untranslated/genetics , RNA-Binding Proteins/metabolism , Sexual Maturation/genetics , Tissue DistributionABSTRACT
The mRNA that encodes the testis-specific protein phosphoglycerate kinase (PGK2) is a long-lived mRNA that is transcribed in meiotic and postmeiotic male germ cells. Pgk2 mRNA is present in germ cells for up to 2 wk before its protein product is detected. Using affinity chromatography with the 3'-UTR of the Pgk2 mRNA, several proteins, including the RNA-binding protein, polypyrimidine tract binding protein 2 (PTBP2), were identified in mouse testis extracts. Coimmunoprecipitation experiments confirmed that PTBP2 binds to Pgk2 mRNA in the testis and RNA gel shifts demonstrated that PTBP2, but not PTBP1, binds to a specific region of the Pgk2 3'-UTR. Recombinant PTBP2 increased the stability of reporter constructs that contained the 3'-UTR Pgk2 sequence element in both testis extracts and transfected HeLa cells. We propose that PTBP2 is a trans-acting factor that helps to stabilize Pgk2 mRNA in male mouse germ cells.
Subject(s)
Isoenzymes/physiology , Nerve Tissue Proteins/metabolism , Phosphoglycerate Kinase/physiology , Polypyrimidine Tract-Binding Protein/metabolism , RNA Stability/physiology , Spermatozoa/metabolism , Animals , Gene Expression Profiling , Germ Cells/metabolism , HeLa Cells , Humans , Isoenzymes/metabolism , Male , Mice , Mice, Inbred Strains , Nerve Tissue Proteins/genetics , Phosphoglycerate Kinase/metabolism , Polypyrimidine Tract-Binding Protein/genetics , Protein Binding , RNA, Messenger/metabolism , RNA-Binding Proteins/metabolism , Regulatory Elements, Transcriptional , Testis/chemistry , Testis/metabolism , TransfectionABSTRACT
MSY2 is a member of the Y-box family of proteins solely expressed in male and female germ cells. In the male, MSY2 serves as a coactivator of transcription by binding to a consensus promoter element present in many germ cell-specific genes. In the nucleus, MSY2 marks specific mRNAs for cytoplasmic storage, stabilization, and suppression of translation. The inactivation of MSY2 by gene targeting leads to spermatogenic arrest and infertility. In testes of mice lacking MSY2, incomplete nuclear condensation is prominent in later-stage spermatids at the time of massive spermatid loss. Because MSY2 interacts with DNA and mRNAs, there are several distinct sites of action, which could be disrupted in mice that lack MSY2, resulting in the arrest of spermatogenesis. To define the molecular cause(s) of the spermatogenic arrest in mice lacking MSY2, transcriptional and posttranscriptional processes were assayed. Transcription, mRNA processing, and mRNA intracellular transport appear normal in the absence of MSY2. However, a redistribution of mRNAs from ribonucleoprotein particles to polysomes and marked decreases were detected for many meiotic and postmeiotic germ cell mRNAs, including the mRNAs encoding the transition proteins and protamines. This suggests that increased mRNA instability is a likely cause of the male infertility in Msy2-null mice.
Subject(s)
DNA-Binding Proteins/physiology , Infertility, Male/genetics , RNA Stability/genetics , RNA-Binding Proteins/physiology , Spermatogenesis/genetics , Animals , Cell Nucleus/ultrastructure , Chromatids/ultrastructure , DNA-Binding Proteins/genetics , Introns , Male , Meiosis/genetics , Mice , Mice, Knockout , Polyribosomes/metabolism , RNA, Messenger/analysis , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Testis/cytology , Testis/metabolism , Transcription, Genetic/geneticsABSTRACT
Transcription of the rat P450c17 gene in Leydig cells requires steroidogenic factor-1 (SF-1) (NR5A1), nerve growth factor-inducible protein B (nurr77), COUP-TF, and SET. The -447/-419 region of this promoter contains two binding sites for orphan nuclear receptors that are required for activation by SF-1, nerve growth factor-inducible protein B, and cAMP. We identified a novel factor, steroidogenic factor-inducer of transcription-2, that binds to this -447/-419 region. We have now purified steroidogenic factor-inducer of transcription-2 from mouse Leydig MA-10 cells and identified it by mass spectrometry as translin, a 27-kDa protein that exerts many functions. By itself, translin cannot activate a P450c17-promoter/reporter construct in HeLa cells; however, translin increased SF-1-stimulated transcription 2-fold, indicating cooperativity between SF-1 and translin. Mutation of both SF-1 binding sites in the -447/-419 sequence eliminated activation by SF-1 and translin. Translin did not augment SF-1-stimulated transcription from all SF-1-responsive elements, suggesting that the activation is specific for the sequence of the SF-1 response element. Gel shift analysis of double- and single-stranded DNA showed that translin binds to single-stranded DNA, but its transcriptional activation is independent of DNA binding. The hinge region of SF-1 is necessary for activation by translin; deletion of hinge amino acids 170-225 in SF-1 eliminates translin's ability to augment SF-1-dependent transcription. A translin-like protein, called translin-associated factor X, can substitute for a translin moiety; translin homomers and translin/translin-associated factor X heteromers activated SF-1-stimulated transcription equally. Thus, we have identified a new factor that works together with SF-1 to augment gene transcription in a DNA-specific fashion.
Subject(s)
DNA-Binding Proteins/physiology , Homeodomain Proteins/physiology , Receptors, Cytoplasmic and Nuclear/physiology , Transcription Factors/physiology , Transcription, Genetic , Animals , Binding Sites , Cell Nucleus/metabolism , Cyclic AMP/metabolism , DNA, Single-Stranded/chemistry , DNA-Binding Proteins/chemistry , HeLa Cells , Homeodomain Proteins/metabolism , Humans , Mass Spectrometry , Mice , Nuclear Receptor Subfamily 4, Group A, Member 1 , Protein Binding , RNA-Binding Proteins , Receptors, Cytoplasmic and Nuclear/chemistry , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Steroid/chemistry , Steroidogenic Factor 1 , Transcription Factors/chemistry , Transcription Factors/metabolism , Transcriptional ActivationABSTRACT
Gametes rely heavily on posttranscriptional control mechanisms to regulate their differentiation. In eggs, maternal mRNAs are stored and selectively activated during development. In the male, transcription ceases during spermiogenesis, necessitating the posttranscriptional regulation of many paternal mRNAs required for spermatozoan assembly and function. To date, most of the testicular mRNAs known to be translationally regulated are initially transcribed in postmeiotic cells. Because protein synthesis occurs on polysomes and translationally inactive mRNAs are sequestered as ribonucleoproteins (RNPs), movement of mRNAs between these fractions is indicative of translational up- and down-regulation. Here, we use microarrays to analyze mRNAs in RNPs and polysomes from testis extracts of prepuberal and adult mice to characterize the translation state of individual mRNAs as spermatogenesis proceeds. Consistent with published reports, many of the translationally delayed postmeiotic mRNAs shift from the RNPs into the polysomes, establishing the validity of this approach. In addition, we detect another 742 mouse testicular transcripts that show dramatic shifts between RNPs and polysomes. One subgroup of 35 genes containing the known, translationally delayed phosphoglycerate kinase 2 (Pgk2) is initially transcribed during meiosis and is translated in later-stage cells. Another subgroup of 82 meiotically expressed genes is translationally down-regulated late in spermatogenesis. This high-throughput approach defines the changing translation patterns of populations of genes as male germ cells differentiate and identifies groups of meiotic transcripts that are translationally up- and down-regulated.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation , Germ Cells/physiology , Meiosis/physiology , Protein Biosynthesis , RNA, Messenger/metabolism , Animals , Cluster Analysis , Down-Regulation , Male , Mice , Oligonucleotide Array Sequence Analysis , Polyribosomes/metabolism , Ribonucleoproteins/metabolism , Testis/physiology , Up-RegulationABSTRACT
Synapse-specific local protein synthesis is thought to be important for neurodevelopment and plasticity and involves neuronal RNA-binding proteins that regulate the transport and translation of dendritically localized transcripts. The best characterized of these RNA-binding proteins is the fragile X mental retardation protein (FMRP). Mutations affecting the expression or function of FMRP cause fragile X syndrome in humans, and targeted deletion of the gene encoding FMRP results in developmental and behavioral alterations in mice. Translin is an RNA-binding protein that regulates mRNA transport and translation in mouse male germ cells and is proposed to play a similar role in neurons. Like FMRP, translin is present in neuronal dendrites, binds dendritically localized RNA, and associates with microtubules and motor proteins. We reported previously the production of viable homozygous translin knock-out mice, which demonstrate altered expression of multiple mRNA transcripts in the brain and mild motor impairments. Here, we report that translin knock-out mice also exhibit sex-specific differences in tests of learning and memory, locomotor activity, anxiety-related behavior, and sensorimotor gating, as well as handling-induced seizures and alterations in monoamine neurotransmitter levels in several forebrain regions. Similar behavioral and neurochemical alterations have been observed in mice lacking FMRP, suggesting that both proteins may act within the same neuronal systems and signaling pathways. Our results in mice indicate that mutations in translin may contribute to fragile X-like syndromes, mental retardation, attention deficit hyperactivity disorder, epilepsy, and autism spectrum disorders in humans.
Subject(s)
Avoidance Learning/physiology , Behavior, Animal/physiology , Biogenic Monoamines/metabolism , Brain/metabolism , DNA-Binding Proteins/deficiency , Neurotransmitter Agents/metabolism , Reaction Time/physiology , Animals , DNA-Binding Proteins/genetics , Female , Male , Maze Learning , Mice , Mice, Inbred C57BL , Mice, Knockout , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Y-box proteins are a well-characterized family of nucleic acid binding proteins that are expressed from bacteria to human. This review will focus on MSY2, a member of the Y-box gene family that is exclusively expressed in male and female germ cells. MSY2 is the mouse ortholog of FRGY2, the Xenopus germ cell-specific protein and the human germ cell protein, Contrin. MSY2 functions as a co-activator of transcription in male germ cells and plays an important role in the translational repression and storage of both paternal and maternal mRNAs in spermatocytes, spermatids and oocytes. Following gene targeting, matings of heterozygotes produce a normal Mendelian ratio with equal numbers of phenotypically normal males and females. However, males and females lacking Msy2 are infertile. In Msy2-null males, spermatogenesis is disrupted in post-meiotic germ cells with many misshapen and multinucleated spermatids. No spermatozoa are found in the epididymis. The germ cell specificity and the critical functions played by this multifunctional DNA- and RNA-binding protein during spermatogenesis make Contrin, the human ortholog of MSY2, an attractive and novel target for male contraception.
Subject(s)
DNA-Binding Proteins/metabolism , Meiosis/genetics , RNA-Binding Proteins/metabolism , Animals , DNA-Binding Proteins/genetics , Female , Gene Deletion , Humans , Male , Mice , RNA-Binding Proteins/genetics , Spermatozoa/cytology , Spermatozoa/metabolism , Transcription Factors/genetics , Transcription Factors/metabolism , Xenopus Proteins/genetics , Xenopus Proteins/metabolismABSTRACT
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.
Subject(s)
DNA-Binding Proteins/genetics , Meiosis , RNA, Messenger/metabolism , RNA, Untranslated/metabolism , Testis/physiology , Animals , Base Sequence , Cerebroside-Sulfatase/genetics , DNA-Binding Proteins/metabolism , Immunoprecipitation , Male , Membrane Proteins/genetics , Mice , Mice, Mutant Strains , Molecular Sequence Data , RNA-Binding Proteins , Receptors, Cell Surface/genetics , Spermatocytes/physiologyABSTRACT
MicroRNAs play important roles in regulating development at both transcriptional and posttranscriptional levels. Here, we report 29 microRNAs from mouse testis that are differentially expressed as the prepubertal testis differentiates to the adult testis. Using computational analyses to identify potential microRNA target mRNAs, we identify several possible male germ cell target mRNAs. One highly conserved sequence in the 3'-untranslated region (UTR) of transition protein 2 (Tnp2) mRNA, a testis-specific and posttranscriptionally regulated mRNA in postmeiotic germ cells, is complementary to Mirn122a. Mirn122a is enriched in late-stage male germ cells and is predominantly on polysomes. Mirn122a, but not another noncomplementary microRNA, inhibits the activity of a luciferase reporter construct containing the 3'-UTR of Tnp2. Site-directed mutations of Mirn122a indicate that base pairing of the 5'-region of Mirn122a to its complementary site in the 3'-UTR of Tnp2 mRNA is essential for the downregulation of luciferase activity. Real-time reverse transcription-polymerase chain reaction and ribonuclease protection assays reveal that the Mirn122a-directed decrease of the Tnp2 reporter gene activity results from mRNA cleavage. We propose that specific microRNAs, such as Mirn122a, could be involved in the posttranscriptional regulation of mRNAs such as Tnp2 in the mammalian testis.
Subject(s)
MicroRNAs/metabolism , Nuclear Proteins/biosynthesis , RNA Processing, Post-Transcriptional/physiology , Animals , Base Sequence , DNA-Binding Proteins , Gene Expression Regulation, Developmental , Male , Mice , MicroRNAs/chemistry , Nuclear Proteins/genetics , Polyribosomes , RNA, Messenger/metabolism , Testis/metabolismABSTRACT
MSY2, a germ-cell-specific member of the Y-box family of DNA-/RNA-binding proteins, is proposed to function as a coactivator of transcription in the nucleus and to stabilize and store maternal and paternal mRNAs in the cytoplasm. In mice lacking Msy2, a normal Mendelian ratio is observed after matings between heterozygotes with equal numbers of phenotypically normal but sterile male and female homozygotes (Msy2-/-). Spermatogenesis is disrupted in postmeiotic null germ cells with many misshapen and multinucleated spermatids, and no spermatozoa are detected in the epididymis. Apoptosis is increased in the testes of homozygotes, and real-time RT-PCR assays reveal large reductions in the mRNA levels of postmeiotic male germ cell mRNAs and smaller reductions of meiotic germ cell transcripts. In females, there is no apparent decrease in either the number of follicles or their morphology in ovaries obtained from 2- and 8-day-old Msy2-/- mice. In contrast, follicle number and progression are reduced in 21-day-old Msy2-/- ovaries. In adult Msy2-/- females, oocyte loss increases, anovulation is observed, and multiple oocyte and follicle defects are seen. Thus, Msy2 represents one of a small number of germ-cell-specific genes whose deletion leads to the disruption of both spermatogenesis and oogenesis.
Subject(s)
DNA-Binding Proteins/metabolism , Infertility , RNA-Binding Proteins/metabolism , Transcription Factors/metabolism , Animals , DNA-Binding Proteins/genetics , Female , Gene Targeting , In Situ Nick-End Labeling , Male , Mice , Mice, Knockout , Oocytes/cytology , Oocytes/physiology , Oogenesis/physiology , Ovary/abnormalities , Ovary/cytology , Ovary/physiology , RNA, Messenger/metabolism , RNA-Binding Proteins/genetics , Spermatogenesis/physiology , Spermatozoa/abnormalities , Spermatozoa/cytology , Spermatozoa/physiology , Testis/cytology , Testis/physiology , Transcription Factors/geneticsABSTRACT
During spermatogenesis, male germ cells temporally synthesize many proteins as they differentiate through meiosis and become spermatozoa. The germ cell Y-box protein, MSY2, constituting approximately 0.7% of total protein in male germ cells, binds to a consensus promoter element, and shows a general lack of RNA-binding specificity. Combining immunoprecipitation and suppressive subtractive hybridization, we identified populations of germ cell mRNAs that are not bound or bound by MSY2. The former population is enriched in cell growth and ubiquitously expressed mRNAs, whereas the latter population is enriched for stored or translationally delayed, male gamete-specific transcripts. Chromatin precipitation assays reveal that most of the MSY2 target mRNAs are transcribed from genes containing the Y-box DNA-binding motif in their promoters. In transgenic mice, mRNAs encoding exogenous GFP are directed or not directed into the MSY2-bound fraction by promoters containing or lacking the Y-box motif, respectively. We propose that MSY2 marks specific mRNAs in the nucleus for cytoplasmic storage, thereby linking transcription and mRNA storage/translational delay in meiotic and postmeiotic male germ cells of the mouse.
Subject(s)
Cytoplasm/physiology , RNA-Binding Proteins/genetics , Spermatozoa/physiology , Animals , Chromatin/physiology , Chromatin/ultrastructure , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Male , Meiosis , Mice , Promoter Regions, Genetic/genetics , Protein Biosynthesis , RNA, Messenger/genetics , RNA-Binding Proteins/metabolism , Recombinant Proteins/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Spermatozoa/cytology , Transcription, GeneticABSTRACT
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.
Subject(s)
DNA-Binding Proteins/metabolism , Kinesins/metabolism , Spermatogenesis/physiology , Spermatozoa/physiology , Animals , DNA, Complementary/genetics , DNA-Binding Proteins/genetics , Kinesins/genetics , Male , Mice , Nerve Tissue Proteins , Protein Binding/genetics , Protein Binding/physiology , Protein Structure, Tertiary/genetics , Protein Structure, Tertiary/physiology , RNA-Binding Proteins , Repressor Proteins , Spermatogenesis/genetics , Two-Hybrid System TechniquesABSTRACT
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.