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.

We previously demonstrated that MSY2, an RNA-binding proteins expressed exclusively in oocytes in females, confers stability to mRNAs during the growth phaseand that CDK1-mediated phosphorylation of MSY2 triggers the transition from mRNAstability to instability. To understand better the function of MSY2 in oocytedevelopment, we characterized further oocytes in which the Msy2 gene has been deleted;this deletion results in infertile females. A decrease of ~30% in the number of oocytesobtained from Msy2-/- mice 22-days of age was observed when compared to wild-type. Msy2-/- -deficient oocytes undergo germinal vesicle breakdown, but display severedeficiencies in spindle formation and chromosome segregation, which likely accounts forthe observed infertility. Oocytes from Msy2 -/- mice are slightly smaller in diameter, butreach the same diameter as oocytes in wild-type mice around day 27. The total amount of mRNA is reduced by about 25% in oocytes from Msy2 -/-mice suggesting that the mRNA is less stable. Moreover, co-injection of a Luc mRNA and Egfp mRNA into wild-type and Msy2-/- oocytes oocytes reveals that Luc mRNAstability, as assayed by measuring luciferase activity, is reduced about three-fold in Msy2-/- oocytes. However, Luc mRNA stability is marked enhanced when Msy2 -/- oocytes areco-injected with Msy2 mRNA. This rescue effect is not observed when the ooctyes are injected with an mRNA encoding a mutant form of Msy2 that cannot bind RNA.Transcription profiling of Msy2 -/- oocytes reveals that ~one third of the transcripts are down-regulated in abundance and the affected genes are implicated in transcription, cell cycle, and protein modification. Interestingly, in contrast to wild-type oocytes that are transcriptionally quiescent, transcription persists in Msy2 -/- oocytes, as assayed by BrUTP incorporation, despite a 4-fold increase in Hdac1 expression. This increase in Hdac1 expression is accompanied by a concomitant decrease in global histoneacetylation using acetylation of histone H4 as a proxy, thereby uncoupling histoneacetylation, which is a mark for permissive chromatin, with the global inhibition of transcription that occurs during oocyte growth. The persistence of transcription in Msy2 -/- oocytes may be linked to the observation that cumulus cells are loosely attached to oocytes obtained from Msy2 -/- mice. Results of these studies provide further evidence for a critical role of MSY2 in stabilizing maternal mRNAs in mouse oocytes during the growth phase. (poster)

In eukaryotic cells, RNA-binding proteins play critical roles in transcription and post-transcriptional events. In the testis, the DNA/RNA-binding protein MSY2 is essential for fertility in both male and female mice. MSY2 selectively binds mRNAs that encode stored or translationally-delayed, male gamete-specific transcripts. In the nucleus MSY2 marks these germ cell mRNAs for cytoplasmic storage, thereby linking transcription and mRNA storage in meiotic and post-meiotic germ cells. Among the many classes of small non-coding RNAs are microRNAs and PIWI-interacting RNAs (piRNAs). Applying a cross-linking and immunoprecipitation procedure (the CLIP assay) with an affinity purified antibody to MSY2, we have recently discovered that the MSY2 protein selectively binds a novel population of small testicular RNAs (MSY-RNAs). MSY-RNAs are ~26-32 nucleotides, often initiate with a 5' adenine, and are expressed throughout germ cell differentiation and in somatic cells. Although most of the MSY-RNAs are derived from annotated genes, a small number (16 of a total of 230 clones sequenced) are piRNAs. piRNAs are an extremely abundant (>500,000 copies) group of non-coding germ cell RNAs that are evolutionarily conserved, but not sequence conserved. All piRNAs are believed to be processed from long transcripts by a MIWI-dependent mechanism. To our surprise, the piRNAs that selectively bind MSY2 are expressed in mice lacking Miwi and the temporal expression of these piRNAs differs greatly from other known piRNAs. In contrast to piRNAs, most MSY-RNAs are derived from widely distributed (non-clustered) sites. MSY-RNAs are present in both nuclei (enriched in chromatin) and cytoplasm and in both ribonucleoprotein particles and polysomes suggesting multiple cellular functions for this new class of small RNA. In summary, we have a identified a new group of MIWI-independent small RNAs that selectively bind to Y-box proteins such as MSY2 and are expressed in both male germ cell and somatic cells. (This research is supported by grant HD44449 from the NIH).

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.

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 ∼75 kDa, is widely expressed in somatic and germ cells where it is primarily nuclear. In addition to the ∼75-kDa KSRP, a ∼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.

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.

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.

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.

Steady state levels of mRNAs in eukaryotic cells are determined by the equilibrium between mRNA synthesis and degradation. In the mammalian testis, expression profiling studies have provided valuable databases of the patterns of gene expression in cell types, but little is known of the mechanisms that stabilize/degrade mRNAs following their transcription. Here we will discuss two distinctively different mechanisms whereby two post-transcriptionally important RNA-binding proteins, MSY2 and PTBP2, stabilize male germ cell mRNAs. MSY2 is a highly conserved and abundant germ cell-specific DNA/RNA-binding protein that functions as a global stabilizer/translational suppressor of mRNAs. Combining immunoprecipitation and suppressive subtractive hybridization, two distinct populations of germ cell mRNAs bound or not by MSY2 were identified. MSY2 bound mRNAs are enriched for stored/translationally-delayed, male gamete-specific transcripts while the non-bound population is enriched for cell growth and ubiquitously expressed mRNAs. Chromatin precipitation assays reveal that most of the MSY2 target mRNAs were transcribed from genes containing a 12nt Y-box DNA binding motif in their promoters. Thus in the nucleus MSY2 binds to a specific promoter DNA sequence before selectively binding to its mRNA thereby linking transcription with cytoplasmic mRNA stabilization. MSY2 is an essential protein for spermatogenesis, because its deletion leads to phenotypically normal, but infertile male and female mice. Although MSY2 has several transcriptional and post-transcriptional functions in germ cells, precocious translation and destabilization of mRNAs causes the male infertility. The days between mRNA appearance and protein synthesis of the glycolytic enzyme, phosphoglycerate kinase 2 (PGK2), represent one of the longest translational delays among the many post-transcriptionally regulated mRNAs in germ cells. Mammals express two functional PGKs. PGK1 is ubiquitously expressed while PGK2 is solely found in male germ cells. During spermatogenesis, PGK2 transcription initiates during meiosis as PGK1 expression ceases and is the sole PGK in post-meiotic germ cells and spermatozoa. Consistent with the discontinuity between mRNA and protein appearance, Pgk2 mRNAs are sequestered as RNPs in meiotic spermatocytes and move onto polysomes in later stage postmeiotic cells. This temporal separation of PGK2 transcription and translation requires long-term Pgk2 mRNA stabilization. PTBP2, a member of the polypyrimidine tract binding protein family, regulates the stability of Pgk2 mRNA by binding to a regulatory element in its 3'UTR. Using RNA affinity chromatography with the 3'UTR of the Pgk2 mRNA and mouse testis extracts, several proteins including PTBP2 were identified. Co-immunoprecipitations established that PTBP2 selectively binds to Pgk2 mRNA, but not to other abundant germ cell mRNAs 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 containing the Pgk2 3'UTR sequence element in both testis extracts and in transfected Hela cells. We conclude that PTBP2 is a trans-acting factor that helps stabilize specific mRNAs such as the Pgk2 mRNA for up to two weeks while MSY2 globally stabilizes a large subpopulation of mRNAs in meiotic and post-meiotic male germ cells. This research was supported by NIH grants HD 04449 and 28832.

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.

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.

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.

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.

cDNA libraries have played a prominent role in developing the extensive database of gene expression in germ cells and somatic cells of the mammalian testis. Differential screening of cDNA libraries has allowed investigators to determine the temporal up- and downregulation of many genes. This chapter discusses how suppressive subtraction hybridization and cDNA sequencing have been used to define populations of messenger RNAs (mRNAs) that selectively bind, or do not bind, to the germ cell-specific Y-box protein, MSY2. MSY2 is an abundant DNA/RNA-binding protein that in vitro binds to all mRNAs, but shows selective binding to a subset of male germ cell mRNAs in cells. This specificity is regulated by MSY2 binding to a conserved sequence in gene promoters, which facilitates MSY2 binding to the transcripts from these promoters in the nucleus and coordinates the transport, storage, and translational suppression of these mRNAs in the cytoplasm.

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.

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.

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.