The gld-1 gene of Caenorhabditis elegans is a germ-line-specific tumor suppressor gene, essential for oogenesis. The gld-1 gene has been cloned and found to encode two proteins that differ by 3 amino acids. The predicted proteins contain an approximately 170-amino-acid region that has been termed the GSG domain (GRP33/Sam68/GLD-1), on the basis of significant similarity between GLD-1, GRP33 from shrimp, and the Src-associated protein Sam68 from mouse (also described as GAPap62 from humans). A conserved structural motif called the KH domain is found within the larger GSG domain, suggesting a biochemical function for GLD-1 protein in binding RNA. The importance of the GSG domain to the function of gld-1 in vivo is revealed by mutations that affect five different conserved GSG domain residues. These include missense mutations in an absolutely conserved residue of the KH domain that eliminate the tumor suppressor function of gld-1 (Jones, 1995).

GLD-1, a putative RNA binding protein, is essential for oocyte development in Caenorhabditis elegans. A gld-1 null mutation abolishes hermaphrodite oogenesis and confers a tumorous germline phenotype in which presumptive female germ cells exit the meiotic pathway and return to the mitotic cell cycle. gld-1(null) germ lines express female-specific, but not male-specific, molecular markers, indicating that gld-1 acts downstream of sexual fate specification to regulate oocyte differentiation. Immunolocalization studies identify GLD-1 as a cytoplasmic germline protein that displays differential accumulation during germline development. Germ cells that are in the mitotic cell cycle contain low levels of GLD-1, most likely a reflection of a nonessential gld-1 function (negative regulation of proliferation in the mitotic germ line) revealed in previous genetic studies. Entry of presumptive oocytes into the meiotic pathway is accompanied by a strong increase in GLD-1 expression/accumulation. GLD-1 levels are high through the pachytene stage but fall to background levels as germ cells exit pachytene and complete oogenesis. The meiotic prophase accumulation pattern is consistent with GLD-1's essential role in oocyte differentiation, which may be to repress until late oogenesis the translation of a subset of maternal RNAs synthesized during early oogenesis, when GLD-1 is absent (Jones, 1996).

Caenorhabditis elegans GLD-1, a KH motif containing RNA-binding protein of the GSG/STAR subfamily, controls diverse aspects of germ line development, suggesting that it may have multiple mRNA targets. An immunoprecipitation/subtractive hybridization/cloning strategy has been used to identify 15 mRNAs that are putative targets of GLD-1 binding and regulation. For one target, the rme-2 yolk receptor mRNA, GLD-1 acts as a translational repressor to spatially restrict RME-2 accumulation, and thus yolk uptake, to late-stage oocytes. GLD-1 binds sequences in both 5' coding and the 3' untranslated region of rme-2 mRNA. Initial characterization of the other 14 targets shows that (1) they are coexpressed with GLD-1; (2) they can have mutant/RNA-mediated interference depletion phenotypes indicating functions in germ line development or as maternal products necessary for early embryogenesis, and (3) GLD-1 may coregulate mRNAs corresponding to functionally redundant subsets of genes within two gene families. Thus, a diverse set of genes have come under GLD-1-mediated regulation to achieve normal germ line development. Previous work identified tra-2 as a GLD-1 target for germ line sex determination. Comparisons of GLD-1-mediated translational control of rme-2 and tra-2 suggests that the mechanisms may differ for distinct target mRNA species (Lee, 2001).

The maternal-effect sterile (MES) proteins are maternally supplied regulators of germline development in Caenorhabditis elegans. In the hermaphrodite progeny from mes mutant mothers, the germline dies during larval development. On the basis of the similarities of MES-2 and MES-6 to known transcriptional regulators and on the basis of the effects of mes mutations on transgene expression in the germline, the MES proteins are predicted to be transcriptional repressors. One of the MES proteins, MES-3, is a novel protein with no recognizable motifs. In this article it is shown that MES-3 is localized in the nuclei of embryos and germ cells, consistent with its predicted role in transcriptional regulation. Its distribution in the germline and in early embryos does not depend on the wild-type functions of the other MES proteins. However, its nuclear localization in midstage embryos and its persistence in the primordial germ cells depends on wild-type MES-2 and MES-6. These results are consistent with biochemical data showing that MES-2, MES-3, and MES-6 associate in a complex in embryos. The distribution of MES-3 in the adult germline is regulated by the translational repressor GLD-1: MES-3 is absent from the region of the germline where GLD-1 is known to be present, MES-3 is overexpressed in the germline of gld-1 mutants, and GLD-1 specifically binds the mes-3 3' untranslated region. Analysis of temperature-shifted mes-3(bn21ts) worms and embryos indicates that MES-3 function is required in the mother's germline and during embryogenesis to ensure subsequent normal germline development. It is proposed that MES-3 acts epigenetically to induce a germline state that is inherited through both meiosis and mitosis and that is essential for survival of the germline (Xu, 2001).

In C. elegans, the Notch receptor GLP-1 is localized within the germline and early embryo by translational control of glp-1 mRNA. RNA elements in the glp-1 3'untranslated region (3' UTR) are necessary for repression of glp-1 translation in germ cells, and for localization of translation to anterior cells of the early embryo. The direct regulators of glp-1 mRNA are not known. A 34 nucleotide region of the glp-1 3' UTR is shown to contain two regulatory elements, an element that represses translation in germ cells and posterior cells of the early embryo, and an element that inhibits repressor activity to promote translation in the embryo. Furthermore, the STAR/KH domain protein GLD-1 binds directly and specifically to the repressor element. Depletion of GLD-1 activity by RNA interference causes loss of endogenous glp-1 mRNA repression in early meiotic germ cells, and in posterior cells of the early embryo. Therefore, GLD-1 is a direct repressor of glp-1 translation at two developmental stages. These results suggest a new function for GLD-1 in regulating early embryonic asymmetry. Furthermore, these observations indicate that precise control of GLD-1 activity by other regulatory factors is important to localize this Notch receptor. Such control contributes to the spatial organization of Notch signaling (Marin, 2003).

Male sex determination in the Caenorhabditis elegans hermaphrodite germline requires translational repression of tra-2 mRNA by the GLD-1 RNA binding protein. fog-2 was cloned by finding that its gene product physically interacts with GLD-1, forming a FOG-2/GLD-1/tra-2 3'untranslated region ternary complex. FOG-2 has an N-terminal F-box and a novel C-terminal domain called FTH. Canonical F-box proteins act as bridging components of the SCF ubiquitin ligase complex; the N-terminal F-box binds a Skp1 homolog, recruiting ubiquination machinery, while a C-terminal protein-protein interaction domain binds a specific substrate for degradation. However, since both fog-2 and gld-1 are necessary for spermatogenesis, FOG-2 cannot target GLD-1 for ubiquitin-mediated degradation. It is proposed that FOG-2 also acts as a bridge, bringing GLD-1 bound to tra-2 mRNA into a multiprotein translational repression complex, thus representing a novel function for an F-box protein. fog-2 is a member of a large, apparently rapidly evolving, C. elegans gene family that has expanded, in part, by local duplications; fog-2 related genes have not been found outside nematodes. fog-2 may have arisen during evolution of self-fertile hermaphroditism from an ancestral female/male species (Clifford, 2000).

Maintenance of the stem cell population in the C. elegans germline requires GLP-1/Notch signaling. This signaling inhibits the accumulation of the KH domain-containing RNA binding protein GLD-1, homolog of Drosophila How. In a genetic screen to identify other genes involved in regulating GLD-1 activity, mutations were identified in the nos-3 gene, the protein product of which is similar to the Drosophila translational regulator Nanos. The data demonstrate that nos-3 promotes GLD-1 accumulation redundantly with gld-2, coding for the catalytic portion of a poly(A) polymerase, and that nos-3 functions genetically downstream or parallel to fbf, an inhibitor of GLD-1 translation. The GLD-1 accumulation pattern is important in controlling the proliferation versus meiotic development decision, with low GLD-1 levels allowing proliferation and increased levels promoting meiotic entry (Hansen, 2003).

This study shows that a major mechanism by which GLP-1/Notch signaling maintains the stem cell population is by inhibiting GLD-1 protein accumulation in the distal end of the germline, thereby restricting its activity to more proximal regions. Not only does low GLD-1 allow proliferation, but high GLD-1 promotes meiosis. The position of the rise in GLD-1 levels determines the size of the stem cell population and the location where germ cells begin meiotic development. nos-3, whose role was identified in a mutant screen, functions redundantly with gld-2 to promote the rise in GLD-1 that is necessary for entry into meiosis. Genetic experiments indicate that repression of GLD-1 accumulation by FBF is acting through nos-3, while regulation of gld-2 in this processes is likely by something other than, or in addition to, FBF. The data suggest a model in which GLP-1 signaling regulates the size of the stem cell population by regulating GLD-1 levels, at least in part, through antagonism between the repressive activity of fbf and the positive activities of nos-3 and gld-2 (Hansen, 2003).

Translational control is an essential mechanism of gene control utilized throughout development, yet the molecular mechanisms underlying translational activation and repression are poorly understood. The translational control of the C. elegans caudal homolog, pal-1, has been investigated and it has been found that GLD-1, a member of the evolutionarily conserved STAR/Maxi-KH domain family, acts through a minimal pal-1 3' UTR element to repress pal-1 translation in the distal germline. Data is provided suggesting that GLD-1 may repress pal-1 translation after initiation. Finally, GLD-1 is shown to repress the distal germline expression of the KH domain protein MEX-3, which was previously shown to repress PAL-1 expression in the proximal germline and which appears specialized to control PAL-1 expression patterns in the embryo. Hence, GLD-1 mediates a developmental switch in the control of PAL-1 repression, allowing MEX-3 to accumulate and take over the task of PAL-1 repression in the proximal germline, where GLD-1 protein levels decline (Mootz, 2004).

GLD-1 is homologous to a sub-family of KH domain proteins known as the GSG or STAR domain family, whose members include the evolutionarily conserved Quaking protein, mammalian Sam68 and SF1 and Drosophila How. The ~200 amino acid STAR domain consists of an enlarged KH RNA-binding domain (maxi-KH domain) flanked by conserved residues on both sides. While the functions of these family members are not well understood, they have been implicated in various aspects of RNA metabolism, including mRNA splicing, nuclear export and translation. Other than GLD-1, only one family member, the mouse Quaking I isoform 6, has thus far been implicated as a translational regulator, and this is based on its ability to repress tra-2 expression when expressed in C. elegans (Mootz, 2004 and references therein).

Multiple in vivo mRNA targets of the maxi-KH/STAR domain protein GLD-1 have been identified by their ability to interact with GLD-1 in cytoplasmic extracts and, for all targets tested thus far, GLD-1 functions as a translational repressor. However, GLD-1 is shown to stabilize the mRNAs of two targets, gna-2 (T23G11.2) and Y75B12B.1. gna-2 mRNA has two upstream open reading frames (uORF), resulting in two premature stop codons. gna-2 mRNA is a naturally occurring mRNA target of nonsense-mediated mRNA decay (NMD), and the binding of GLD-1 protects gna-2 mRNA from NMD, likely by repressing translation of the uORFs. Therefore, gna-2 mRNA comes under two posttranscriptional controls: (1) translation regulation by a specific translational repressor, GLD-1, and (2) uORF elicited regulation, mainly through NMD. As a result, these two posttranscriptional controls together provide precise temporal and spatial control of gene expression. Consistent with this novel mode of regulation, when GLD-1 mRNA targets acquire premature stop codon mutations, GLD-1 protects them from NMD. Analysis of several mRNA targets containing premature stop codons suggests that in translation repression, GLD-1 either represses ribosome assembly on the target mRNA, or subsequent ribosome elongation to the premature stop codon (Lee, 2004).

Regulation of gene expression is central to most cellular processes and occurs at multiple steps, including transcription, splicing, nuclear export, localization, stability, and translation of mature mRNAs. Germ-line and early embryonic development are particularly dependent on posttranscriptional control of maternal mRNAs for temporal/spatial regulation of gene expression because the genome is transcriptionally silent from late meiotic prophase (diakinesis), through the meiotic divisions/fertilization and into early embryogenesis. Expression of several maternal mRNAs is regulated by more than one posttranscriptional mechanism. For example, during Drosophila oogenesis and early embryogenesis, the combination of two posttranscriptional controls, localization and translational control, precisely regulates the expression of oscar, bicoid, and nanos mRNAs. In most cases, cis-acting elements in the 5'-or 3'-untranslated regions (UTRs) of mRNAs mediate their posttranscriptional regulation. When a specific mRNA harbors more than one cis-acting element, and therefore is under more than one posttranscriptional control, its expression is determined combinatorially; this results in precise temporal and spatial control of gene expression (Lee, 2004 and references therein).

One important cis-acting element in the 5'-UTR of several eukaryotic mRNAs is an upstream open reading frame (uORF). uORFs often control the efficiency of translation of the downstream main ORF, primarily by altering the activity of the ribosome. After translation initiation factors bind at the 5'-cap, recruit the small 40S ribosome subunit, and scan to the AUG of the uORF (uAUG), the scanning initiation complex sometimes fails to recognize the uAUG, and proceeds to the downstream AUG (leaky scanning). However, more often the complex recognizes the uAUG and initiates translation, resulting in the production of nascent peptides that can sometimes interfere with translation termination; as a result, the ribosome stalls at the stop codon (ribosome stalling). After the ribosome completes translation of the uORF, it usually dissociates from the mRNA, resulting in the failure to translate the downstream ORF. Alternatively, the 40S subunit can remain associated with the mRNA and resume scanning (reinitiation). In general, reinitiation is inefficient and only occurs after translation of very short uORFs. In addition, several studies have shown that uORFs also affect mRNA stability through nonsense-mediated mRNA decay (NMD), because the stop codon of the uORF can be regarded as a premature stop codon. Several examples of mRNAs with one or more uORFs indicate that uORFs can elicit multiple modes of regulation, such as leaky scanning and termination-dependent decay for YAP2 mRNA, which together exert precise control over the amount of protein synthesized (Lee, 2004 and references therein).

NMD can be viewed as a surveillance system that prevents the production of truncated proteins by degrading mRNAs harboring premature stop codons due to mutations, errors induced during transcription or splicing, or mRNAs transcribed from out-of-frame gene rearrangements in the vertebrate immune system. In addition, NMD also contributes to the fine tuning of normal gene expression by degrading specific mRNAs that have naturally occurring premature stop codons -- for example, uORF-containing mRNAs. NMD factors have been identified genetically in yeast and Caenorhabditis elegans. Three yeast (UPF1-3) and seven C. elegans (smg-1-smg-7, suppressor with morphogenic defects on genitalia) genes have been shown to play essential roles in NMD. The UPF1/2/3 proteins (SMG-2/SMG-3/SMG-4 in C. elegans) are conserved from yeast to humans, whereas C. elegans SMG-1, SMG-5, and SMG-7 have orthologs in higher eukaryotes but not in yeast (Lee, 2004).

Many studies have provided a working model of NMD. NMD requires translation initiation and procession of the ribosome to the premature stop codon, whereupon factors are recruited to degrade the mRNA. Current data support a model in which mammalian UPF3 is loaded onto the mRNA during splicing by interacting with an exon junction complex (EJC), while UPF2 joins the complex soon after mRNA export. UPF2 and UPF3 are displaced by ribosomes as they traverse the mRNA during the first round of translation. Translation termination at a premature stop codon recruits UPF1, probably by interacting with the eRF1-eRF3 translation termination complex. The incomplete removal of UPF2 and/or UPF3 from downstream mRNA sequences results in the assembly of the NMD complex, consisting minimally of UPF1, UPF2, and UPF3. mRNAs that have premature stop codons, and therefore allow the assembly of this complex, are targeted for rapid degradation (Lee, 2004).

GLD-1 is a member of a family of proteins, including human/mouse Quaking, SAM68, and Drosophila Held out wings, all of which share an ~200-amino acid region of similarity called the GSG or STAR domain. The conserved region contains a maxi-KH RNA-binding motif that differs from the canonical FMR1/Nova KH motif by the addition of three loops that are conserved only among GSG/STAR proteins and also contains conserved regions N-terminal and C-terminal to the maxi-KH motif. The GSG/STAR domain is essential for in vivo function, because missense mutations in nine different conserved residues in this domain alter or eliminate gld-1 function. GLD-1 has multiple functions during C. elegans germ cell development, including the regulation of meiotic prophase progression of female germ cells, the mitotic versus meiotic switch, and the promotion of the male fate in the hermaphrodite germ line. GLD-1 is exclusively expressed in the germ line and is localized in the cytoplasm of the distal region. The level of GLD-1 is very low in the distal mitotic zone. As germ cells begin meiotic development in the transition zone, the level of GLD-1 increases to its maximum level and stays high through the pachytene stage in the distal region. Levels decrease at the end of pachytene and become undetectable in developing oocytes in the proximal region. The nonhomogeneous distribution of GLD-1, and its role as a cytoplasmic RNA-binding protein that functions in multiple aspects of germ cell development, indicate that GLD-1 functions to spatially restrict the translation of multiple maternal mRNAs (Lee, 2004).

Multiple in vivo mRNA targets of GLD-1 have been identified that coimmunoprecipitate with GLD-1 from cytoplasmic extracts. These mRNA targets are preferentially expressed in the germ line and, as expected, several of them exhibit essential functions in oocyte differentiation and early embryogenesis. For at least one target, rme-2 mRNA, GLD-1 functions as a translational repressor. RME-2 protein is not expressed in pachytene-stage germ cells in the distal region of wild-type germ line where GLD-1 is abundant in the cytoplasm, but is expressed in developing oocytes in the proximal region where GLD-1 is absent. RME-2 is misexpressed in the distal region of gld-1-null germ line. Even though RME-2 protein is not expressed in the distal region of wild-type germ lines, rme-2 mRNA is expressed and accumulates (Lee, 2004).

GLD-1 targets can be subjected to an additional control mechanism at the posttranscriptional level. The results of this study demonstrate the existence of a novel mechanism in which the expression of at least one GLD-1 mRNA target is precisely controlled by the combination of uORF-mediated NMD together with GLD-1-dependent translational repression. Consistent with this mechanism, translational repression by GLD-1 is shown to protect mRNA targets that contain nonsense mutations from NMD (Lee, 2004).

The isolation and early developmental expression of a zebrafish homolog of qkI is described. The zebrafish quaking cDNA, zqk, exhibits striking conservation with qkI across the coding region, accompanied by a unique 123 nucleotide insertion sequence. Maternal and zygotic zqk transcripts are ubiquitously distributed during cleavage and blastula periods, and then accumulate in the dorsal midline of the body trunk during gastrulation. During segmentation and pharyngula periods, zqk transcripts are expressed in the neural tissue of the head region, and in the paraxial mesoderm of the body trunk. Subsequently, they diminish until the hatching period, when they are expressed only in the cardiac sac and pectoral finbuds. The zqk transcript is alternatively spliced with the transcript containing a 123 nucleotide additional segment localized in neural tissue in the head region, but not in the paraxial mesoderm in the body trunk. The data suggest that the quaking gene family originated in the mesoderm and evolved to become expressed in the nervous system in lower vertebrates. The insertion of the 123 nucleotide sequence could be related to the acquisition of a neural function for the gene (Tanaka, 1997).

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. cDNAs encoding the Xenopus quaking homolog (Xqua) have been isolated and characterized, and an almost complete human quaking sequence was assembled 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, RNA-binding activity has been demonstrated 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 (Zorn, 1997a).

Xenopus homolog of quaking, Xqua, has been isolated and the sequence has been shown to be highly conserved through evolution. The quaking protein expressed during early embryogenesis, pXqua357, can bind RNA in vitro; the regions of the protein that are essential for RNA binding have been mapped. pXqua can form homodimers and dimerization may be required for RNA binding. Oocyte injection experiments show that pXqua357 is located in both the nucleus and cytoplasm. In the Xenopus embryo, Xqua is first expressed during gastrulation in the organizer region and its derivative, the notochord. In later stage embryos, Xqua is expressed in a number of mesodermal and neural tissues. Disruption of normal Xqua function, by overexpression of a dominant inhibitory form of the protein, blocks notochord differentiation. Xqua function appears to be required for the accumulation of important mRNAs such as Xnot, Xbra, and gsc. These results indicate an essential role for the quaking RNA-binding protein during early vertebrate embryogenesis (Zorn, 1997b).

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 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 (Ebersole, 1996).

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 (Hardy, 1996).

Qk1 is a member of the KH domain family of proteins that includes Sam68, GRP33, GLD-1, SF1, and Who/How. These family members are RNA binding proteins that contain an extended KH domain embedded in a larger domain called the GSG (for GRP33-Sam68-GLD-1) domain. An ethylnitrosourea-induced point mutation in the Qk1 GSG domain alters glutamic acid 48 to a glycine and is known to be embryonically lethal in mice. The Qk1 GSG domain mediates RNA binding and Qk1 self-association. By using in situ chemical cross-linking studies, it has been shown that the Qk1 proteins exist as homodimers in vivo. The Qk1 self-association region maps to amino acids 18 to 57, a region predicted to form coiled coils. Alteration of glutamic acid 48 to glycine (E->G) in the Qk1 GSG domain (producing protein Qk1:EG) abolishes self-association but has no effect on the RNA binding activity. The expression of Qk1 or Qk1:-EG in NIH 3T3 cells induces cell death by apoptosis. Approximately 90% of the remaining transfected cells are apoptotic 48 h after transfection. Qk1:EG is consistently more potent at inducing apoptosis than is wild-type Qk1. These results suggest that the mouse quaking lethality occurs due to the absence of Qk1 self-association mediated by the GSG domain (Chen, 1998).

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 ~200-amino acid STAR domain, which contains a single extended heteronuclear ribonucleoprotein K homolog domain flanked by two domains called QUA1 and QUA2. QKI isoforms can 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 Quaking isoforms have different carboxyl termini and different subcellular localization. GFP fusion protein was used to identify 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, this motif has been named 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, it was 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 (Wu, 1999).

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. Both in vitro and in vivo, 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, these data support the hypothesis that other STAR family members may also be TGE-dependent translational regulators (Saccomanno, 1999).

Quakingviable (qk^v) is a well known dysmyelination mutation. Recently, the genetic lesion of qk^v has been defined as a deletion 5' to the qkI gene, which results in the severe reduction of the qkI-encoded QKI RNA-binding proteins in myelin-producing cells. However, no comprehensive model has been proposed regarding how the lack of QKI leads to dysmyelination. It is hypothesized that QKI binds to myelin protein mRNAs, and the lack of QKI causes posttranscriptional misregulation, which in turn leads to the loss of the corresponding myelin proteins. To test this hypothesis, an RNase protection assay was developed to directly measure the mRNA isoforms encoding the myelin basic proteins (MBPs) in the brain. Isoform-preferential destabilization of MBP mRNAs in the cytoplasm is responsible for the reduced MBPs in the qk^v/qk^v brain during early myelination. In addition, markedly reduced MBP mRNAs are detected in the qk^v/qk^v myelin fraction with concomitant accumulation of MBP mRNAs associated with membrane-free polyribosomes. Presumably, the impaired localization of MBP mRNAs to the myelin membrane may cause insufficient incorporation of the newly synthesized MBPs into the myelin sheath. Interactions are observed between QKI and MBP mRNAs, and removing MBP 3'UTR significantly reduces QKI-binding. Taken together, these observations suggest that misregulation at multiple posttranscriptional steps is responsible for the severe reduction of MBPs in qk^v dysmyelination, presumably because of the lack of interactions between MBP mRNAs and the QKI RNA-binding proteins (Li, 2000).

quaking viable mice have myelination defects and develop a characteristic tremor 10 d after birth. The quaking gene encodes at least five alternatively spliced QUAKING (QKI) isoforms that differ in their C-terminal 8-30-amino-acid sequence. The reasons for the existence of the different QKI isoforms and their function are unknown. One QKI isoform, QKI-7, can induce apoptosis in fibroblasts and primary rat oligodendrocytes. Heterodimerization of the QKI isoforms results in the nuclear translocation of QKI-7 and the suppression of apoptosis. The unique C-terminal 14 amino acids of QKI-7 confers the ability to induce apoptosis to heterologous proteins such as the green fluorescent protein and a QKI-related protein, Caenorhabditis elegansGLD-1. Thus, the unique C-terminal sequences of QKI-7 may function as a life-or-death 'sensor' that monitors the balance between the alternatively spliced QKI isoforms. Moreover, these findings suggest that nuclear translocation is a novel mechanism of inactivating apoptotic inducers (Pilotte, 2001).

The genetic lesion of quakingviable (qk^v) causes diminished expression of the QKI RNA-binding protein in myelin producing cells. Consequently, several structural myelin proteins are severely reduced. Among these affected proteins, the reduction of the myelin basic protein (MBP) results from post-transcriptional abnormalities of the MBP mRNA, presumably due to the lack of interactions with QKI. However, whether this is the common mechanism for reduced expression of other myelin proteins in qk^v dysmyelination remains unclear. Distinct molecular mechanisms underlie the reduction of MBP and the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in qk^v dysmyelination. MBP transcripts bind QKI strongly and are markedly reduced in the qk^v/qk^v oligodendrocytes in which QKI is almost completely lost. In contrast, CNP transcripts bind QKI weakly and are only slightly affected by the lack of QKI. None the less, CNP proteins are severely reduced in the qk^v/qk^v brain. Since CNP transcripts are predominantly associated with translating polyribosomes, diminished CNP expression in qk^v dysmyelination is unlikely to be due to translational failures, but more likely results from accelerated protein degradation (Zhang, 2001).