The quaking I-5 protein (QKI-5) has a novel nuclear localization signal and shuttles between the nucleus and the cytoplasm.

The mouse quaking (qk) gene is essential in both myelination and early embryogenesis. Its product, QKI, is an RNA-binding protein belonging to a growing protein family called STAR (signal transduction and activator of RNA). All members have an approximately 200-amino acid STAR domain, which contains a single extended heteronuclear ribonucleoprotein K homologue domain flanked by two domains called QUA1 and QUA2. We found that QKI isoforms could associate with each other, while one of the lethal mutations qkI(kt4) with a single amino acid change in QUA1 domain, leads to a loss of QKI self-interaction. This suggests that the QUA1 domain is responsible for QKI dimerization. Three QKI isoforms have different carboxyl termini and different subcellular localization. Here, using GFP fusion protein, we identified a 7-amino acid novel nuclear localization sequence in the carboxyl terminus of QKI-5, which is conserved in a subclass of STAR proteins containing SAM68 and ETLE/T-STAR. Thus, we name this motif STAR-NLS. In addition, the effects of active transcription, RNA-binding and self-interaction on QKI-5 localization were analyzed. Furthermore, using an interspecies heterokaryon assay, we found that QKI-5, but not another STAR protein ETLE, shuttles between the nucleus and the cytoplasm, which suggests that QKI-5 functions in both cell compartments.

The STAR protein QKI-6 is a translational repressor.

The signal transduction and activation of RNA (STAR) family of RNA-binding proteins, whose members are evolutionarily conserved from yeast to humans, are important for a number of developmental decisions. For example, in the mouse, quaking proteins (QKI-5, QKI-6, and QKI-7) are essential for embryogenesis and myelination, whereas a closely related protein in Caenorhabditis elegans, germline defective-1 (GLD-1), is necessary for germ-line development. Recently, GLD-1 was found to be a translational repressor that acts through regulatory elements, called TGEs (for tra-2 and GLI elements), present in the 3' untranslated region of the sex-determining gene tra-2. This gene promotes female development, and repression of tra-2 translation by TGEs is necessary for the male cell fates. The finding that GLD-1 inhibits tra-2 translation raises the possibility that other STAR family members act by a similar mechanism to control gene activity. Here we demonstrate, both in vitro and in vivo, that QKI-6 functions in the same manner as GLD-1 and can specifically bind to TGEs to repress translation of reporter constructs containing TGEs. In addition, expression of QKI-6 in C. elegans wild-type hermaphrodites or in hermaphrodites that are partially masculinized by a loss-of-function mutation in the sex-determining gene tra-3 results in masculinization of somatic tissues, consistent with QKI-6 repressing the activity of tra-2. These results strongly suggest that QKI-6 may control gene activity by operating through TGEs to regulate translation. In addition, our data support the hypothesis that other STAR family members may also be TGE-dependent translational regulators.

Genomic organization and expression analysis of the mouse qkI locus.

qkI, encoding a KH domain-containing RNA binding protein, has been isolated as a candidate gene for the mouse neurological mutation quaking. Here, we describe detailed studies on its genomic structure and expression pattern. We isolated approximately 1 Mb of genomic region containing the quaking locus and determined its genomic organization. The qkI locus contains at least 9 exons spanning approximately 65 kb of DNA. It gives rise to six distinct transcripts encoding, theoretically, five different protein isoforms. Exons 1 through 4 are shared by all the transcripts, whereas coding exons and two distinct 3'-UTRs downstream to the exon 4 are differentially utilized. One isoform has a truncated KH domain and may act as an antagonist to the others. These findings and identification of a single transcription initiation site suggest that differential expression of each transcript is regulated by alternative splicing. Expression of each alternative transcript and protein product was also examined. Two types of transcripts, 5 kb-A and B, are most abundant in the brain of newborn mice and are gradually downregulated thereafter. In contrast, the other three messages, 6 kb, 7 kb-A and B, increase as myelination proceeds and peak at 2 weeks of age, corresponding to the most active stage of myelination. Although the qkI messages and their products are abundant in brain and heart, a lower level of expression was found in various other tissues tested. Alternative transcripts that share the same 3'-UTR showed very similar expression patterns, suggesting a regulatory role of the 3'-UTRs in qkI gene expression.

T-STAR/ETOILE: a novel relative of SAM68 that interacts with an RNA-binding protein implicated in spermatogenesis.

RBM is an RNA-binding protein encoded on the Y chromosome in mammals and is expressed only in the nuclei of male germ cells. Genetic evidence from infertile men implicates it in spermatogenesis, but its function is unknown. Of a number of potential partners for RBM identified by a yeast two-hybrid screen with testis cDNA, the most frequent isolates encoded a novel RNA-binding protein, termed T-STAR, that is closely related to SAM68, an Src-associated protein of unknown function. The mouse homologue was also cloned and designated étoile. It mapped to chromosome 15, while T-STAR mapped to the syntenic region on human chromosome 8. T-STAR/étoile is expressed primarily in the testis; in rat germ cells, the expression of both T-STAR/étoile and SAM68 is regulated during meiosis. Transfection of T-STAR/étoile fused with green fluorescent protein into HeLa cells caused an accumulation of protein in a novel compartment of the nucleus, adjacent to the nucleolus but distinct from the peri-nucleolar compartment. RBM and other hnRNP G family members are candidate downstream targets for regulation by T-STAR/ETOILE and SAM68.

Loss of function of the tuberous sclerosis 2 tumor suppressor gene results in embryonic lethality characterized by disrupted neuroepithelial growth and development.

Germline defects in the tuberous sclerosis 2 (TSC2) tumor suppressor gene predispose humans and rats to benign and malignant lesions in a variety of tissues. The brain is among the most profoundly affected organs in tuberous sclerosis (TSC) patients and is the site of development of the cortical tubers for which the hereditary syndrome is named. A spontaneous germline inactivation of the Tsc2 locus has been described in an animal model, the Eker rat. We report that the homozygous state of this mutation (Tsc2(Ek/Ek)) was lethal in mid-gestation (the equivalent of mouse E9.5-E13.5), when Tsc2 mRNA was highly expressed in embryonic neuroepithelium. During this period homozygous mutant Eker embryos lacking functional Tsc2 gene product, tuberin, displayed dysraphia and papillary overgrowth of the neuroepithelium, indicating that loss of tuberin disrupted the normal development of this tissue. Interestingly, there was significant intraspecies variability in the penetrance of cranial abnormalities in mutant embryos: the Long-Evans strain Tsc2(Ek/Ek) embryos displayed these defects whereas the Fisher 344 homozygous mutant embryos had normal-appearing neuroepithelium. Taken together, our data indicate that the Tsc2 gene participates in normal brain development and suggest the inactivation of this gene may have similar functional consequences in both mature and embryonic brain.

Mouse Brachyury the Second (T2) is a gene next to classical T and a candidate gene for tct.

The mouse Brachyury the Second (T2) gene is 15 kb away from classical Brachyury (T). A mutation in T2 disrupts notochord development, pointing to the existence of a second T/t complex gene involved in axis development. T2 encodes a novel protein that is disrupted by an insertion in T2(Bob) mice. Sequence analysis of T2 from several t haplotypes shows that they all share the same changed stop codon, and, thus, T2 is a candidate gene for the t complex tail interaction factor. T1, T2, and the unlinked t-int are distinct and unrelated loci, and mutations in these genes do not complement one another genetically. Either their products interact in the same pathway during the genesis of the embryonic axis, or the T/t region itself is truly complex.

Spermatid perinuclear ribonucleic acid-binding protein binds microtubules in vitro and associates with abnormal manchettes in vivo in mice.

Spermatid perinuclear RNA-binding protein (SPNR) is a microtubule-associated RNA-binding protein that localizes to the manchette in developing spermatids. The RNA target of SPNR in vivo is unknown, although we have previously suggested the possibility that SPNR is involved in the translational activation of the protamine 1 mRNA in elongated spermatids. To increase our understanding of SPNR's association with the manchette, we sought to determine SPNR's subcellular localization in several mouse mutants that show reduced fertility or sterility and that have structurally abnormal manchettes. We show here that despite the highly abnormal manchettes and microtubule aggregates formed in azh, hop-sterile, tw2, and tw8 mutants, SPNR remains associated with the manchettes. Localization of SPNR to the abnormal manchettes suggests that SPNR is tightly bound to the manchette. SPNR could bind manchette microtubules directly, or it could bind indirectly via an interaction with a microtubule-associated protein (MAP). We sought to determine whether SPNR binds microtubules in vitro, and if so, whether it requires a MAP. We show by Western analysis that the endogenous SPNR protein can be pelleted with murine testis microtubules in a taxol-dependent manner in vitro. A recombinant version of SPNR produced in bacteria can also be pelleted with testis microtubules, as well as microtubules polymerized from purified bovine brain tubulin, an association that is salt-sensitive. These results suggest that SPNR, in addition to its function as an RNA-binding protein, is also a bona fide MAP.

Remarkable sequence conservation of transcripts encoding amphibian and mammalian homologues of quaking, a KH domain RNA-binding protein.

Mutations in the mouse quaking locus can result in two different types of developmental phenotypes: (1) a deficiency of myelin in the central nervous system that is accompanied by a characteristic tremor, or (2) embryonic lethality around day 9 of gestation. A quaking candidate gene (qkI) that encodes a KH motif protein has recently been identified. We have isolated and characterized cDNAs encoding the Xenopus quaking homologue (Xqua) and also assembled an almost complete human quaking sequence from expressed sequence tags. Sequence comparisons show that the amphibian and mammalian quaking transcripts exhibit striking conservation, both within the coding region and, unexpectedly, in the 3' UTR. Two Xqua transcripts 5 kb and 5.5 kb in length are differentially expressed in the Xenopus embryo, with the 5 kb transcript being detected as early as the gastrula stage of development. Using an in vitro assay, we have demonstrated RNA-binding activity for quaking protein encoded by the 5 kb transcript. Overall, the high sequence conservation of quaking sequences suggests an important conserved function in vertebrate development, probably in the regulation of RNA metabolism.

STAR, a gene family involved in signal transduction and activation of RNA.

A new subfamily of KH-domain-containing RNA-binding proteins is encoded by genes that are conserved from yeast to humans. Mutations with interesting developmental phenotypes have been identified in Caenorhabditis elegans, Drosophila and mouse. It is hypothesized that these bifunctional proteins provide a rich source of interesting molecular information about development and define a new cellular pathway that links signal transduction directly to RNA metabolism.

Neural cell type-specific expression of QKI proteins is altered in quakingviable mutant mice.

qkI, a newly cloned gene lying immediately proximal to the deletion in the quakingviable mutation, is transcribed into three messages of 5, 6, and 7 kb. Antibodies raised to the unique carboxy peptides of the resulting QKI proteins reveal that, in the nervous system, all three QKI proteins are expressed strongly in myelin-forming cells and also in astrocytes. Interestingly, individual isoforms show distinct intracellular distributions: QKI-6 and QKI-7 are localized to perikaryal cytoplasm, whereas QKI-5 invariably is restricted to the nucleus, consistent with the predicted role of QKI as an RNA-binding protein. In quakingviable mutants, which display severe dysmyelination, QKI-6 and QKI-7 are absent exclusively from myelin-forming cells. By contrast, QKI-5 is absent only in oligodendrocytes of severely affected tracts. These observations implicate QKI proteins as regulators of myelination and reveal key insights into the mechanisms of dysmyelination in the quakingviable mutant.

Oct-4 regulates alternative platelet-derived growth factor alpha receptor gene promoter in human embryonal carcinoma cells.

Expression of the platelet-derived growth factor alpha-receptor (PDGFalphaR) gene is tightly controlled in mammalian embryogenesis. A well established model system to study human embryogenesis is the embryonal carcinoma cell line Tera2. We have shown previously that retinoic acid-differentiated Tera2 cells express two PDGFalphaR transcripts of 6.4 kilobase pairs (kb) (encoding the full-length receptor) and 3.0 kb, respectively, whereas in contrast, undifferentiated Tera2 cells express PDFGalphaR transcripts of 1.5 kb and 5.0 kb. Here we show that this switch in PDGFalphaR expression pattern during differentiation of Tera2 cells results from alternative promoter use. In undifferentiated cells, a second promoter is used, which is located in intron 12 of the PDGFalphaR gene. Functional analysis shows that this promoter contains a consensus octamer motif, which can be bound by the POU domain transcription factor Oct-4. Oct-4 is expressed in undifferentiated Tera2 cells but not in retinoic acid-induced differentiated cells. Mutation of the octamer motif decreases promoter activity, while ectopic expression of Oct-4 in differentiated Tera2 cells specifically enhances the activity of this PDGFalphaR promoter. Therefore, we suggest that an important aspect in the maintenance of the undifferentiated state of human embryonal carcinoma cells results from Oct-4 expression, which thereupon activates this PDGFalphaR promoter.

The quaking gene product necessary in embryogenesis and myelination combines features of RNA binding and signal transduction proteins.

The mouse quaking gene, essential for nervous system myelination and survival of the early embryo has been positionally cloned. Its sequence implies that the locus encodes a multifunctional gene used in a specific set of developing tissues to unite signal transduction with some aspect of RNA metabolism. The quaking(viable) (qkv) mutation has one class of messages truncated by a deletion. An independent ENU-induced mutation has a nonconservative amino acid change in one of two newly identified domains that are conserved from the C. elegans gld-1 tumour suppressor gene to the human Src-associated protein Sam68. The size and conservation of the quaking gene family implies that the pathway defined by this mutation may have broad relevance for rapid conveyance of extracellular information directly to primary gene transcripts.

Is there a Brachyury the Second? Analysis of a transgenic mutation involved in notochord maintenance in mice.

A new phenotype mapping to the t-complex, which is designated Brachyury the Second (T2), is characterized by a slightly shortened tail in heterozygotes and homozygous failure to form an organized notochord with subsequent abnormal development of posterior somites and neural tube. The phenotype of T2 superficially resembles that of Brachyury; however, there are several important differences. Brachyury homozygotes fail to make posterior somites, notochord, floor plate, and a placental connection, resulting in death by 10.5 days of development. In contrast, T2 homozygotes make posterior somites, scattered notochord cells, and floorplate and achieve an allantoic connection. However, despite making a maternal connection, T2 homozygotes cease development at E11.5 and die soon after. We have cloned and analyzed the transgene insertion site, which maps within 100 kb of the Brachyury gene, but does not seem to physically interrupt nor affect transcription from that locus. The existence of a second gene mapping near Brachyury and affecting the same developmental processes was alluded to over 50 years ago and has been debated ever since. An embryological description of T2 is presented, as is a discussion of the implications of a single, larger Brachyury locus versus two closely linked genes coordinately regulating axial development.

Tctex2: a sperm tail surface protein mapping to the t-complex.

Transmission ratio distortion (TRD) in mouse t-haplotypes remains the most significant example of meiotic drive in vertebrates. While the underlying mechanism that fuels it is still mysterious, TRD is clearly a complex multigene phenomenon. The characterization of Tctex2 (t-complex testis expressed 2) shows it to be one of several candidates for involvement in TRD. Tctex2 maps to the t-complex and encodes a membrane-associated protein found exclusively on the sperm tail. The t-haplotype form of Tctex2 is aberrant in both the level of its expression and its primary amino acid sequence, but is nonetheless translated and transported to its normal location. The multiple amino acid changes in the t-form make it extremely unlikely that it can function normally and, since it is found on sperm tails, suggest that it may actively interfere with the development of normal gamete function in males. The possible role of Tctex2 in t-complex transmission ratio distortion and sterility is discussed.

Identification of a germ-cell-specific transcriptional repressor in the promoter of Tctex-1.

The Tctex-1 gene family maps to the t complex of the mouse and consists of four copies on chromosome 17 in both wild-type and t-haplotypes. Tctex-1 mRNA is eightfold overexpressed in male and female germ cells in t-haplotype compound heterozygotes (tx/ty). In order to determine the cause of this aberrant expression and the role of this gene family in spermatogenesis and oogenesis it was subjected to extensive molecular analysis. We find that Tctex-1 protein is present in sperm tails and oocytes and that it is present at equal levels in wild-type and t-haplotype testis. Surprisingly, the excess message in t-haplotypes is not translated. Sequence analysis of the gene family reveals that one copy in t-haplotypes has a mutated start codon. This same copy is deleted for a protein-binding motif in its promoter. This motif, GIM (Germ cell Inhibitory Motif) has strong homology to the Xenopus AP-2-binding site but does not appear to be a binding site for mammalian AP-2. A factor(s) present in testis and ovary, but absent in other mouse tissues binds specifically to this site. Transfection assays using Tctex-1 promoter constructs suggest that GIM functions as a transcriptional repressor. The possible role of Tctex-1 in t complex transmission ratio distortion and sterility is discussed.