InteractiveFly: GeneBrief

held out wings : Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References


Gene name - held out wings

Synonyms - struthio

Cytological map position - 93F

Function - putative RNA-binding protein

Keywords - wing, muscle, heart

Symbol - how

FlyBase ID: FBgn0017397

Genetic map position -

Classification - KH motif

Cellular location - nuclear



NCBI links: Precomputed BLAST | Entrez Gene
BIOLOGICAL OVERVIEW

held out wings (how), also known as struthio, was first identifed using the P-transposable element enhancer trap screen in studies conducted by the Jan, Perrimon, and Scott laboratories (Perrimon, 1991). A P-element is a small, autonomously movable fragment of DNA, a transposable element, capable of being inserted at any place in the organism's genome. Such an insertion can cause a mutation, and multiple inbred lines are generated, each of which carry a single P-element insertion. Some how P-element insertion mutants are viable when combined with a deficiency that deletes the site of P-element insertion, although one line displays a weak adult wings held-out phenotype. Insertion mutants vary in the severity of their how aberrations, from complete loss-of-function to partial loss-of-function. how mutants fall into distinct classes based on their lethal phases during development, forming an allelic series (from the developmentally earliest lethals to those who reach some phase of adult viability (Baehrecke, 1997).

Animals lacking how function die late during embryogenesis, possessing defects that seem to occur sometime between myoblast fusion and muscle cell attachment. A lethal mutant dies with the most posterior region of the cuticle arrested above the dorsal surface, presumably cause by failure to complete germ band retraction. howe44 mutants have normally differentiated and fused myotubules; with variable expressivity, the myotubes of some embryos appear to migrate, but are disorganized and don't possess the proper pattern of attachment, while the myotubules of other mutant embryos show no signs of migration, and actually appear to have fewer myoblasts. Partial loss-of-function results in lethality during the metamorphosis from larva to adult. Most howr17 homozygotes die during metamorphosis, with their heads stuck inside their thoraxes. A small number of howr17 homozygotes survive to adulthood (escape) but do not fold their wings properly and, thus, possess the phenotype for which this gene is named. These flies also have blisters on their wings (Baehrecke, 1997).

Flies that apparently possess mutant clones of cells of the stru1A122 mutation exhibit wing blisters. These blisters are fluid filled in wings of newly eclosed flies, eventually collapsing as the flies mature. Veins and interveins within these blisters appear to be normal. Wing blisters that result from a disruption in the process of cell adhesion between the two wing epithelial layers are also seen in mutant alleles of the Drosophila PS integrin subunit genes (see Myospheroid). Due to the similarity in the wing blister phenotype, flies with mutant clones were examined for two other postembryonic phenotypes associated with mutations in the PS integrin genes, namely, disruption of eye morphogenesis and impairment of flight. Although no defects were found in eye structure, homozygous clones in adults result in impaired flight. This defect could not be ascribed to the presence of wing blisters, as some flies with no apparent wing defects exhibited flight impairment (Lo, 1997).

In studies by both Lo (1997) and Zaffran (1997), no defects in how mutants could be found in muscle structure. But additional behavioral defects were found by Zaffran. Observations focussed on the heart. Even though the development of the heart appears normal, the heart rate is considerably slower than in wild-type animals. Heart pulsations in Drosophila start 16 hours after fertilization; their number increases to 60 per minute in 3rd instar larvae. In mutant how18 embryos, the heart contractions are of very small amplitude, appearing as small palpitations rather than as the constriction movements that occur in the wild-type cardiac myoendothelium. The hearts of mutant embryos begin to contract on time, according to the normal developmental schedule and the rate progressively reaches 20-30 pulsations per minute. However, rates never exceed this value and decrease later. Interestingly, the heart region of the dorsal vessel, the lumen, is very narrow, indicating either that it is blocked in a contracted state or that the complete differentiation of the heart is not effective in how18 mutants.

Baehrecke (1997) reports defects in the arrangement of muscle cell precursors that are likely to serve as a basis for understanding the behavioral defects in how mutants. how defects are similar to the aberrations exhibited by previously identified mutants in genes whose products function in the migration and attachment of somatic muscle cells. The myotubes of animals that lack the function of myospheroid, for example, migrate to appropriate attachment sites, but fail to maintain attachment. Like how mutants, myospheroid mutants exhibit a cuticle defect that is caused by incomplete germ band retraction. However, how mutant muscle fibers are perturbed earlier than the muscles of myospheroid mutants and appear to be defective in migration rather than attachment. This migration defect is more similar to the phenotype of stripe and derailed mutants, both of which form myotubes that exhibit defects in orientation during migration (Baehrecke, 1997).

how is transcribed in muscles and in the epidermal locations where muscles attach. This is similar to the expression of derailed, which is expressed in the same cell populations. These genes differ from the position-specific integrins, however, which are expressed in complemetary patterns in either muscle or muscle attachment site cells. how and derailed also differ from stripe expression, which is restricted to epidermal muscle attachemnt sites. When adult muscle development is occurring during metamorphosis, both integrins and stripe, like how, are expressed in a similar population of cells. It is difficult to predict how genes that encode DNA or RNA binding proteins like stripe and how may function in muscle cell migration. RNA binding proteins encoded by genes like how could have various functions, including RNA transport, stability and splicing. Future studies of how will determine how a gene that encodes an RNA binding protein functions in muscle development (Baehrecke, 1997).

Two isoforms of the Drosophila RNA binding protein, How, act in opposing directions to regulate tendon cell differentiation

Differential RNA metabolism regulates a wide array of developmental processes. A mechanism is described that controls the transition from premature Drosophila tendon precursors into mature muscle-bound tendon cells. This mechanism is based on the opposing activities of two isoforms of the RNA binding protein How. While the isoform How(L) is a negative regulator of Stripe, the key modulator of tendon cell differentiation, How(S) isoform elevates Stripe levels, thereby releasing the differentiation arrest induced by How(L). The opposing activities of the How isoforms are manifested by differential rates of mRNA degradation of the target Stripe mRNA. This mechanism is conserved, as the mammalian RNA binding Quaking proteins may similarly affect the levels of Krox20, a regulator of Schwann cell maturation (Nabel-Rosen, 2002).

RNA binding proteins of the Signal Transduction and Activation of RNA (STAR) family may regulate gene expression at various levels, e.g., at the level of nuclear export of the target mRNA, at the level of mRNA stability, and at the translation level. The How proteins appear to exert their activity through their effect on mRNA stability. How(L) appears to induce rapid degradation of the target RNA, an activity that is tightly coupled to its nuclear retention and depends on the presence of the nuclear retention signal that is conserved in QKI-5. It has been suggested that How(L) may prevent nuclear export of its target mRNA. Indeed, in embryos overexpressing How(L), the mRNA of gfp-sr3'UTR is occasionally detected in the nucleus. Presently, it is not possible to determine whether the primary effect of How(L) is retention of the target mRNA in the nucleus followed by degradation of the target mRNA or vice versa. How(S) increases the stability of the same target RNA. The fact that How(S) is present both in the nucleus and in the cytoplasm raises the possibility that the association of How(S) with its target RNA during and following its nuclear export leads to mRNA stabilization. A number of RNA binding proteins possess both nuclear and cytoplasmic functions, e.g., proteins that affect both mRNA export and mRNA stability. Similarly, How proteins may affect both nuclear-cytoplasm shuttling and mRNA stability. The differential association of each of the How proteins with distinct protein partners presumably leads to their opposing effects on mRNA stability. A possible mechanism for the counteraction effect of How(S) may arise from its association with How(L), eliminating the repression by How(L). Indeed, How(S) and How(L) are coprecipitated from Schneider cells following their cotransfection together with the gfp-sr3'UTR (Nabel-Rosen, 2002).

A recent report suggests that a sequence (TGE) in the 3'UTR of C. elegans tra-2, essential for Gld-1 binding, mediates deadenylation and poly(A)-dependent translation repression. Poly(A) deadenylation may also lead to mRNA degradation. Since a sequence motif that is partially related to TGE is also present in the stripe and krox20 3'UTR, degradation of the target mRNA by How(L) may be based on a similar mechanism. Recently, the two cytoplasmic hnRNPs, K and E1, have been shown to inhibit translation of lipoxigenase mRNA by preventing its attachment to the 60S ribosomal unit (Ostareck, 2001). The possibility cannot be excluded that How(S), in addition to its positive effect on mRNA stability, may also facilitate translation efficacy (Nabel-Rosen, 2002).

The results suggest that the relative amount of How(L) and How(S) during different stages of embryonic or adult development regulate the switch between the premature and mature state of differentiation of tendon cells. In early stages of embryonic development, How(L) prevails, Stripe expression is downregulated, and differentiation is arrested. In later stages of embryonic development, How(S) is upregulated, overriding How(L) repression and facilitating Stripe expression. The difference in Stripe mRNA levels may be further enhanced by Stripe transcriptional autoregulation. What could be the mechanism that regulates the relative levels of How proteins during tendon cell maturation? Northern analysis suggests that the total levels of How(L) mRNA are low throughout embryonic development, relative to those of How(S) mRNA. At the protein level, the proportion of the two proteins is inverted; How(S) protein levels are low and increase only during late stages of embryonic development. This suggests that How(S) is posttranscriptionally regulated. Indeed, transgenic flies carrying How(S) with its unique 3'UTR exhibit almost undetectable levels of How(S) protein following induction by the gal4 driver. When this 3'UTR is deleted, the expression levels of How(S) are significantly higher. The expression of How(S) appears to be elevated by Vein-mediated activation of the ->F receptor pathway in tendon cells following muscle-tendon association. The molecular link between ->F receptor activation and the upregulation of How(S) has yet to be elucidated. A recent report suggesting that ERK phosphorylation of hnRNP-K drives cytoplasmic accumulation of hnRNP-K (Habelhah, 2001) may be of relevance if, similarly to hnRNP-K, How(S) undergoes ERK-dependent phosphorylation (Nabel-Rosen, 2002).

The mechanism described here for the activity of How proteins appears to be conserved in evolution. QKI proteins regulate the expression of target genes in opposing directions. QKI-5 represses while QKI-6 and QKI-7 facilitate the expression of target RNAs. Moreover it appears that krox20 mRNA is an endogenous target for QKI activity. It is suggested that QKI proteins regulate the transition from premature to mature Schwann cells by repressing or elevating the levels of krox20 as well as those of additional target mRNAs. Thus, it is likely that the relative proportions of the inhibitor (QKI-5) and facilitators (QKI-6 and QKI-7) determine the state of Schwann cell differentiation. It is already known that QKI-5 is highly expressed in premature Schwann cells throughout embryonic development and that QKI-6 and QKI-7 are upregulated during Schwann cell maturation, consistent with the possibility that the latter are positive regulators of Schwann cell maturation. Krox20 has been recently shown to induce a wide array of genes, many of which are essential for myelination. Loss of krox20 is associated with human myelinopathies. If QKI proteins modulate the level of Krox20 during Schwann cells maturation, then reduction in QKI levels may result in abnormal Krox20 levels during Schwann cell myelination. Indeed, reduced levels of QKI (presumably all the isoforms) observed in qkiv mice lead to severe myelination defects (Nabel-Rosen, 2002).

A recent study in mice has identified the mRNA of myelin basic protein (MBP) as a target for Quaking activity. In adult qkiv mice, the levels of MBP are reduced, as a result of destablization of MBP mRNA. The fact that, at the adult stage, QKI-6 and QKI-7 [equivalent to How(S)] are the predominant isoforms suggests that their activity, like that of How(S), is required for stabilization of MBP mRNA. Apparently, the levels of QKI-5 in this mutant are high enough to carry on embryonic development. It would be essential in future studies to analyze the effect of each of the QKI isoforms on target mRNAs in Schwann cells (Nabel-Rosen, 2002).

Are there other QKI homologs in Drosophila? Out of ten proteins related to quaking recently described in the Drosophila genome (Di Fruscio, 1998; Fyrberg, 1998; Lasko, 2000), how shows the highest similarity to Quaking and therefore may represent its true ortholog (Nabel-Rosen, 2002).


REGULATION

Transcriptional Regulation

The early expression of How protein is apparently under the control of twist. A direct role for twist in the mesodermal expression of how is further suggested by the existence of four putative Twist binding-sites in a 80 base pair intronic fragment. Also consistent with this hypothesis is the fact that How is expressed in the precursor cells of adult muscles that re-express twist. Early expression is not modified in either snail or tinman mutants. The expression is not affected in a mef2 mutant in which late differentiation stages of muscular cells is altered (Zaffran, 1997).

In order to test if how is transcriptionally regulated by ecdysone (see Ecdysone receptor), third larval instar tissues were isolated before the increase in ecdysone titer. They were cultured for 8 hours, and a physiologically high titer of ecdysone was added to cultures for varying periods of time. RNA was extracted from the tissues, electorphoresed, transferred to a membrane, and hybridized with a radiolabelled how probe. how is induced by ecdysone in cultured organs (Baehrecke, 1997).

Muscle-dependent maturation of tendon cells is induced by post-transcriptional regulation of stripeA

Terminal differentiation of single cells selected from a group of equivalent precursors may be random, or may be regulated by external signals. In the Drosophila embryo, maturation of a single tendon cell from a field of competent precursors is triggered by muscle-dependent signaling. The transcription factor Stripe induces both the precursor cell phenotype, as well as the terminal differentiation of muscle-bound tendons. The mechanism by which Stripe activates these distinct differentiation programs remained unclear. This study demonstrates that each differentiation state is associated with a distinct Stripe isoform and that the Stripe isoforms direct different transcriptional outputs. Importantly, the transition to the mature differentiation state is triggered post-transcriptionally by enhanced production of the stripeA splice variant, which is typical of the tendon mature state. This elevation is mediated by the RNA-binding protein How(S), with levels sensitive to muscle-dependent signals. In how mutant embryos the expression of StripeA is significantly reduced, while overexpression of How(S) enhances StripeA protein as well as mRNA levels in embryos. Analysis of the expression of a stripeA minigene in S-2 cells suggests that this elevation may be due to enhanced splicing of stripeA. Consistently, stripeA mRNA is specifically reduced in embryos mutant for the splicing factor Crooked neck (Crn), which physically interacts with How(S). Thus, a mechanism is generated by which tendon cell terminal differentiation is maintained and reinforced by the approaching muscle (Volohonsky, 2007).

This study demonstrates the involvement of post-transcriptional control in a cell-differentiation process that must be coupled to muscle-tendon interaction. Terminal differentiation of tendons involves a major reorganization of the microtubule and actin networks. Such processes are presumably not compatible with embryonic morphogenetic movements such as germ band retraction. Thus, it is essential to spatially and temporally restrict differentiation to single muscle-bound tendon cells. Indeed, the results show that premature overexpression of StripeA in the entire ectoderm leads to severe defects in germ band retraction (Volohonsky, 2007).

Stripe mediates both the determination of precursor cells as well as their maturation and ability to undergo specific temporal and spatial regulation. The current findings suggest both negative and positive feedback loops, based on post-transcriptional regulation of stripe splice variants that on one hand maintain non-bound tendon cells at the precursor state, and on the other hand enable irreversible differentiation of muscle-bound tendons (Volohonsky, 2007).

Whereas some tissue differentiation processes (e.g. tracheal development) initiate upon the expression of a key transcription factor, which autoregulates its own expression, thus leading to a unidirectional differentiation route, other cells (e.g. cells in the proneural region) go through an intermediate stage of a field of competent precursors, in which only additional local interactions lead to irreversible differentiation. Maturation of tendon cells follows the latter path, although the selection mechanism is based on regulation at the post-transcription level (Volohonsky, 2007).

The following model is used to explain the transition between the two phases of tendon cell development: the initial expression of stripeB is induced by segment polarity-dependent signals. StripeB defines a set of tendon precursor cells. StripeB then reinforces its own expression and in addition induces How(L) expression, which in turn suppresses stripeB mRNA levels, thus keeping StripeB levels constant throughout embryonic development. This is supported by experiments that show that StripeB overexpression leads to elevation in How(L) and in StripeB itself. Following myotube extension and adhesion to a tendon precursor cell, How(S) levels are elevated in the muscle-adherent tendon cells, presumably due to EGFR activation. How(S) associates with the splicing factor Crn and the complex shuttles into the nucleus, where it binds to stripeA intronic sequences and elevates its mRNA levels, by enhancing its splicing and maintaining the stability of the spliced mRNA. The resulting muscle-bound tendon cell expresses high StripeA levels, which further drive the expression of genes required for terminal tendon differentiation (e.g. shot, how), as inferred from StripeA overexpression experiments. This regulatory mechanism couples muscle binding and tendon cell maturation, while preventing differentiation of additional, non-bound, precursors (Volohonsky, 2007).

RNA-binding proteins can function as adaptor units promoting the assembly of large protein complexes that control the various aspects of RNA metabolism. How, together with Quaking and GLD-1, belongs to the Star family of RNA-binding proteins, the members of which often regulate more than one facet of RNA metabolism. For example, GLD-1 has been suggested to regulate mRNA stability as well as translation of some of its targets. Similarly Quaking controls mRNA stability as well as RNA splicing, and possibly also mRNA nuclear export and localization. It appears that How proteins also exhibit a wide range of activities on RNA metabolism. While the effect of How(L) and How(S) on stripe mRNA stability has been demonstrated previously, this study suggests that How(S) has an additional activity in regulating the splicing of stripeA. Consistent with this study, How has been identified in a dsRNA-based screen for alternative splicing regulators, as a protein required for specific splicing of exons within two out of five tested genes, paralytic (exons A/I), and Dscam (exon 4), in S-2 cells. Previous studies suggested that the ability of How proteins to stabilize stripe mRNA is mediated by the 3' UTR of stripe. However, the splicing of stripeA appears to be regulated by its specific intronic sequences (Volohonsky, 2007).

By contrast to How(L), which is localized specifically in the nucleus, How(S) is distributed both in the nucleus and the cytoplasm. However, when How(S) is retained in the nucleus by the addition of an NLS sequence, it loses its effect on the mRNA levels of its target. What could be the molecular explanation for the involvement of How(S) in splicing? It is suggested that How(S) binds to a cytoplasmic splicing factor and recruits it to the nucleus, where it is targeted to bind stripeA-specific intronic sequences. This may enhance the splicing of stripeA-specific exons. A candidate splicing factor is Crn. Crn is a general, well-conserved splicing factor that is expressed by a wide range of cell types and is distributed both in the nucleus and the cytoplasm. In a parallel study, it was demonstrated that crn and how mutants exhibit closely related phenotypes, affecting glial cell maturation. Importantly, both Crn and How(S) proteins [but not How(S)-NLS] coprecipitate from S-2 cell extracts, indicating that both proteins are associated in a common protein complex in the cytoplasm (Edenfeld, 2006). In addition, when Crn is myristoylated and transfected into S-2 cells together with How(S), How(S) is relocated to the membrane (Edenfeld, 2006). Furthermore, in crn mutants StripeA, but not StripeB, levels are reduced, and this is reflected in the reduction of Shot levels (Volohonsky, 2007).

These results support a model in which How(S) interacts with Crn in the cell cytoplasm, shuttles into the nucleus and facilitates stripeA splicing, and possibly mRNA stability, leading to StripeA protein elevation. A similar mechanism may operate in the Quaking-dependent facilitation of myelin-associated glycoprotein splicing (Volohonsky, 2007).

In summary, a molecular mechanism has been described that is based on post-transcriptional control, by which cell differentiation is induced and maintained by local interactions with neighboring cells (Volohonsky, 2007).

Dissection of the target specificity of the RNA-binding protein HOW reveals dpp mRNA as a novel HOW target

Regulation of RNA metabolism plays a major role in controlling gene expression during developmental processes. The Drosophila RNA-binding protein Held out wing (HOW), regulates an array of developmental processes in embryonic and adult growth. The primary sequence and secondary structural requirements for the HOW response element (HRE) has been characterized; this site (ACUAA) is necessary and sufficient for HOW binding. Based on this analysis, the Drosophila TGFß homolog, dpp, was identified as a novel direct target for HOW negative regulation in the wing imaginal disc. The binding of the repressor isoform HOW(L) to the dpp 3' untranslated region (UTR) leads to a reduction of GFP-dpp3'UTR reporter levels in S-2 cells, in an HRE site-dependent manner. Moreover, co-expression of HOW(L) in the wing imaginal disc with a dpp-GFP fusion construct led to a reduction in DPP-GFP levels in a dpp-3'UTR-dependent manner. Conversely, a reduction of the endogenous levels of HOW by targeted expression of HOW-specific double-stranded RNA led to a corresponding elevation in dpp mRNA level in the wing imaginal disc. Thus, by characterizing the RNA sequences that bind HOW, a novel aspect has been demonstrated of regulation, at the mRNA level, of Drosophila DPP (Israeli, 2007).

It has been shown that HOW binds directly to the 3'UTR of stripe. To characterize the HOW-binding sites further, the stripe 3'UTR 1.2 kb sequence was truncated into smaller fragments, which were individually transcribed in vitro and labeled with biotin. These fragments were tested for HOW binding by adding in vitro-translated HOW tagged with hemagglutinin (HA) to the biotin-labeled RNA followed by precipitation of the RNA complexes using avidin-conjugated magnetic beads. The presence of HOW on the beads was then tested by western blot analysis using anti-HA antibodies. As a control for non-specific binding, a mutant HOW variant (HOWm) was used, that carries a mis-sense mutation in the KH domain exchanging arginine at position 185 to cysteine, mimicking the severe loss-of-function howe44 allele. HOWm does not exhibit RNA-binding activity. This analysis allowed selection of two HOW-binding fragments (a and b) in which the sequence ACUAA, which was similar, but not identical, to the GLD-1 hexanucleotide-binding site in tra-2, was identified. In fragment a, there are three repeats of this sequence, and fragment b contains one such sequence (Israeli, 2007).

It is concluded that the sequence ACUAA represents the primary HRE. Importantly, one of the HRE sequences (at position 766) is conserved in the 3'UTR of stripe in Drosophila pseudoobscura. Moreover, three repeats of the pentamer AAUAA (which also binds HOW, but to a lesser extent) were identied that are conserved between the two Drosophila species. Thus the HOW-binding site NA(C>A)UAA closely resembles that of STAR proteins from other species, although it is not identical. The binding of HOW was studied in the context of the entire stripe 3'UTR, and it was demonstrated that deletion of these four sites indeed abrogates the responsiveness of the stripe 3'UTR to HOW (Israeli, 2007).

Because a pentanucleotide sequence would be relatively abundant within the 3'UTRs of many RNAs, it was suspected that additional restrictions might exist in addition to the primary sequence ACUAA. Analysis of the distinct HOW-binding sites in the stripe 3'UTR using the Mfold program showed that high-affinity binding for HOW occurs when the binding site (ACUAA) is included within a single-stranded loop. However, secondary-structure predictions of large RNA fragments (larger than 30-40 nucleotides) using the Mfold program resulted in numerous alternatives. To test whether a loop secondary structure is essential for the binding of HOW, HRE-containing loops of distinct sizes were constructed, fused to the 3' end of the stripe 3'UTR fragment (1-225), which does not bind HOW. It was found that single-stranded loops that are larger than 12 nucleotides and contain a single HRE site exhibit significant binding, whereas loops smaller than 12 nucleotides did not exhibit specific binding to HOW. Presumably, these loops are too small to allow this binding (Israeli, 2007).

Structural studies helped identify a novel HOW target, namely dpp mRNA, in the wing imaginal disc. Normally, the repressor isoform of HOW, HOW(L), reduces dpp mRNA levels in the wing imaginal disc and in the pupal wing, leading to reduced DPP protein levels during the establishment of the anteroposterior axis, and later during wing vein formation. Presumably, in the absence of HOW(L), higher DPP levels at the source would alter the overall shape of the DPP gradient, thus expanding the Spalt expression domain. The phenotype of ectopic veins obtained by continuous expression of HOW(L) dsRNA in the pupal wings supports an additional role for HOW(L) in repressing dpp mRNA at later stages of wing development (Israeli, 2007).

The sensitivity of the embryo to DPP levels has been demonstrated by the DPP haplo-insufficient phenotype. This sensitivity is also exhibited in the wing imaginal disc by the observation that endogenous dpp can be replaced by UAS-GFP-dpp driven by dpp-gal4 only at low temperatures (16°C or 19°C), at which the Gal4 protein is significantly less active. Because the responsiveness of the cells to DPP levels is highly sensitive, it is necessary to tightly regulate the levels of DPP protein; for example, by constitutive reduction of its mRNA levels in DPP-secreting cells by the HOW(L) protein (Israeli, 2007).

Cell divisions in the Drosophila embryonic mesoderm are repressed via posttranscriptional regulation of string/cdc25 by HOW

Cell-cycle progression is tightly regulated during embryonic development. In the Drosophila early embryo, the levels of String/Cdc25 define the precise timing and sites of cell divisions. However, cell-cycle progression is arrested in the mesoderm of gastrulating embryos despite a positive transcriptional string/cdc25 activation provided by the mesoderm-specific action of Twist. Whereas String/Cdc25 is negatively regulated by Tribbles in the mesoderm at these embryonic stages, the factor(s) controlling string/cdc25 mRNA levels has yet to be elucidated. This study shows that the repressor isoform of the Drosophila RNA binding protein Held Out Wing [HOW(L)] is required to inhibit mesodermal cell division during gastrulation. Embryos mutant for how exhibit an excess of cell divisions, leading to delayed mesoderm invagination. The levels of the mitotic activator string/cdc25 mRNA in these embryos were significantly elevated. Protein-RNA precipitation experiments show that HOW(L) binds string/cdc25 mRNA. Overexpression of HOW(L) in Schneider cells reduces specifically the steady-state mRNA levels of a gfp reporter fused to string/cdc25 untranslated region (3'UTR). These results suggest that in wild-type embryos, string/cdc25 mRNA levels are downregulated by the repressor isoform HOW(L), which binds directly to string/cdc25 mRNA and regulates its degradation. Thus, this study proposes a novel posttranscriptional mechanism controlling cell-cycle progression in the Drosophila embryo (Nabel-Rosen, 2005; full text of article).

String/Cdc25 is a limiting factor that controls cell-cycle progression in early embryonic stages after cellularization. Both string/cdc25 mRNA and String/Cdc25 protein are extremely unstable (T1/2 < 15 min). The instability of the mRNA and protein allows for a sensitive response of String/Cdc25 levels to transcriptional regulation by various transcription factors operating in pattern formation in the embryo. It has been reported that, in addition to the time of initiation of string/cdc25 transcription, accumulation of string/cdc25 mRNA is slower in mitotic domain 10 (MD10) than in MD2. This is consistent with lower mRNA levels detected in MD10 in relation to MD2 in wild-type embryos. This study provides a molecular basis for string/cdc25 mRNA instability. In situ hybridization with string antisense probe as well as RT-PCR experiments demonstrated that, in how mutant embryos, string/cdc25 is upregulated. Moreover, protein-RNA binding experiments showed a direct binding between HOW and string RNA, and in Schneider cells a gfp-string3'UTR reporter mRNA is specifically degraded in the presence of HOW(L). Collectively, these experiments are consistent with HOW(L) being the major factor responsible for string instability in the early embryo (Nabel-Rosen, 2005).

A consensus RNA binding site, (U>A/C/G)ACUAA, has been recently described for the binding of the STAR protein Gld-1. The same sequence has also been characterized in as being a consensus RNA binding site for HOW. Importantly, this sequence, GACUAA, is present in the string 3'UTR. These results are consistent with the idea that HOW binds the relaxed consensus sequence described for Gld-1, which is also present in string 3′UTR. Interestingly, this sequence appears also in the C. elegans cdc25/string homolog, suggesting that Gld-1 (similarly to HOW in Drosophila) may control cdc25/string in C. elegans (Nabel-Rosen, 2005).

The arrest of cell-cycle progression in the invaginating mesoderm must be transient because immediately after the invagination process the cells undergo a round of cell division. Thus, String/Cdc25 protein levels must be downregulated to a narrow time window to enable mesoderm invagination. This time frame may be achieved in the following manner: Twist, a regulator of mesoderm fate, activates the transcription of string/cdc25 and HOW. Maternal HOW, as well as zygotic HOW shown previously to be downstream of Twist, compromises string/cdc25 mRNA levels, and Tribbles (which requires Twist and Snail to perform its activity) reduces String/Cdc25 protein levels at this stage. Thus, in parallel to string/cdc25 transcriptional activation, Twist provides a double safe mechanism that silences string at the mRNA via HOW activity and String protein via Tribbles activity in MD10. The activity of both HOW and Tribbles should enable the eventual accumulation of String/Cdc25 protein at the end of the invagination process to allow cell-cycle progression at this stage. Therefore, both HOW's and Tribbles's inhibitory effect may not be highly efficient. This, together with the constitutive transcriptional activation of string by Twist, may lead to the eventual accumulation of string mRNA and protein levels, overcoming the negative control imposed by HOW and Tribbles. Alternatively, String accumulation may be caused by a more direct inhibition of both HOW and Tribbles activities, possibly by signaling pathways that operate in the mesoderm after its invagination (Nabel-Rosen, 2005).

Maternal HOW appears to reduce string mRNA levels in the lateral ectoderm in stage-5 and -6 embryos in addition to MD10. It is therefore possible that the extra cell divisions detected in these regions may have an additional indirect effect on mesoderm invagination (Nabel-Rosen, 2005).

It is instructive to ask whether HOW regulates additional processes during mesoderm development. Although zygotic how mutants do not exhibit mesoderm defects until late developmental stages, the how germline clone embryos do show significant mesoderm aberrations. The entire somatic muscle pattern of these embryos is severely disrupted, presumably owing to accumulation of defects. At this stage, it is impossible to distinguish between primary and secondary effects induced by the complete lack of HOW. The muscle defects detected in how germline clone embryos suggest that HOW has a broader function in the mesoderm and that it may regulate the levels of an array of essential genes necessary for appropriate mesoderm development. The identification of such genes should elucidate the full regulatory range of HOW activity (Nabel-Rosen, 2005).

Finally, regulation at the level of mRNA metabolism by STAR family proteins has been shown to occur in several developmental systems, for example, C. elegans gld-1 and mammalian quaking. These proteins exhibit a wide range of activities, affecting RNA splicing, mRNA nuclear export, mRNA stability, and possibly others. The advantage of such regulation is the ability to respond rapidly to external signals by controlling the mRNA levels of an array of target genes. The synchronization between muscle-cell differentiation and cell-cycle progression may be based on the activities of both HOW and Tribbles, but the molecular link between both processes has yet to be elucidated (Nabel-Rosen, 2005).

Post-transcriptional repression of the Drosophila midkine and pleiotrophin homolog miple by HOW is essential for correct mesoderm spreading

The even spreading of mesoderm cells in the Drosophila embryo is essential for its proper patterning by ectodermally derived signals. In how germline clone embryos, defects in mesoderm spreading lead to a partial loss of dorsal mesoderm derivatives. HOW is an RNA-binding protein that is thought to regulate diverse mRNA targets. To identify direct HOW targets, a series of selection methods were implemented on mRNAs whose levels were elevated in how germline clone embryos during the stage of mesoderm spreading. Four mRNAs were found to be specifically elevated in the mesoderm of how germline clone embryos and to exhibit specific binding to HOW via their 3' UTRs. Importantly, overexpression of three of these genes phenocopied the mesoderm-spreading phenotype of how germline clone embryos. Further analysis showed that overexpressing one of these genes, miple, a Drosophila midkine and pleiotrophin heparin-binding growth factor, in the mesoderm leads to abnormal scattered MAPK activation, a phenotype that might explain the abnormal mesoderm spreading. In addition, the number of EVE-positive cells, which are responsive to receptor tyrosine kinase (RTK) signaling, was increased following Miple overexpression in the mesoderm and appeared to be dependent on Heartless function. In summary, these analysis suggests that HOW downregulates the levels of a number of mRNA species in the mesoderm in order to enable proper mesoderm spreading during early embryogenesis (Toledano-Katchalski, 2007).

Previous analysis of how germline clones suggested that HOW is essential for correct mesoderm spreading over the ectoderm (Nabel-Rosen, 2005), a process that is required for the spatial patterning of the mesoderm layer by ectoderm-derived signals. This phenotype could not result from the earlier effect of extra cell division during gastrulation detected in the how germline clone embryos, because mesoderm invagination and gastrulation were eventually completed in these embryos. Furthermore, mesoderm spreading was unaffected in tribbles (trbl) mutant embryos, which exhibit a similar defect of extra cell divisions during gastrulation that leads to delayed and unsynchronized mesoderm invagination (Toledano-Katchalski, 2007).

Regulation of mesoderm-specific mRNA levels by HOW might contribute to the spatial and temporal control of gene expression during mesoderm spreading. The genome analysis was designed to identify mRNAs whose levels might be directly controlled by the repressor isoform HOW(L) in the mesoderm. Such targets should be normally repressed to enable even spreading of the mesoderm. Three out of the four HOW targets identified in this screen, namely falten, CG31638 and LAP (CG2520) are contributed maternally, and therefore HOW(L)-dependent repression in the mesoderm might be essential for reducing their levels in this tissue to enable proper mesoderm spreading. This scenario is supported by the defective mesoderm spreading induced by overexpression of falten and CG31638. Miple does not appear to be maternally contributed according to expression data and in situ analysis. It is not clear, however, which transcription factor is responsible for miple induction. Because miple mRNA was detected in mesoderm derivatives at stage 11, it might be induced by mesoderm-specific transcription factors such as Twist, MEF2 and/or Tinman, which are expressed in the mesoderm during spreading. In that case, to abrogate the effects of Miple, it would be necessary to block miple expression during mesoderm spreading. These data suggest that this is the role of HOW(L), because in its absence, miple mRNA is significantly elevated in the mesoderm. Thus, HOW(L) in the mesoderm of gastrulating embryos is necessary to reduce maternal mRNA expression and, in addition, to reduce the levels of gene products whose expression is not compatible with early mesoderm development, but might be required shortly after the process of mesoderm spreading has been completed. Thus, HOW(L) is essential to enable temporal morphogenetic processes in the mesoderm during its spreading over the ectoderm (Toledano-Katchalski, 2007).

Miple was further analyzed because its vertebrate homologs, midkine and pleiotropin, are involved in cell migration and are associated with receptor tyrosine kinase (RTK) signaling (Stoica, 2002). Therefore, its downregulation by HOW(L) might contribute to the restricted dorsal activation of the HTL-dependent signaling during mesoderm spreading. Moreover, the putative heparin-binding motif of Miple could affect the affinity of the HTL ligands to the HTL receptor, thereby modulating the strength of HTL-dependent signaling (Toledano-Katchalski, 2007).

Indeed, the findings suggest that downregulation of Miple levels in the mesoderm is essential for correct mesoderm spreading, because Miple overexpression leads to impaired mesoderm spreading. The disordered pattern of MAPK phosphorylation (detected by anti-dpERK antibody) observed following Miple overexpression might be the primary cause for the mesoderm spreading defect. In wild-type embryos, MAPK activation is detected only at the most dorsal cells of the spreading mesodermal cells. The mechanism by which this spatial MAPK activation is achieved is not clear. It has been suggested that MAPK activation takes place only in cells that directly contact the ectoderm. In that case, Miple might trigger prolonged mesoderm-ectoderm cell contacts and this could delay mesoderm spreading. Indirect evidence, especially the observation that overexpression of an activated form of HTL does not lead to an ectopic dpERK signal in the entire mesoderm, led to the suggestion that a constitutive inhibitory input of MAPK activation is present in mesoderm cells (Wilson, 2005). This inhibitory activity was suggested to be overcome only in cells that form close contact with the ectoderm. It is unlikely that the role of Miple is to counteract this inhibitory signal, because overexpression of Miple has an effect not only on MAPK activation in early mesoderm spreading but also on the late HTL-dependent signaling in the dorsal EVE-positive cells, in which this inhibitory signal has not been implicated. Therefore the possibility is favored that Miple enhances HTL signaling, and that this enhancement is reflected by MAPK activation in both early and later stages of mesoderm development (Toledano-Katchalski, 2007).

The elevation of the dpERK signal detected following overexpression of Miple might be mediated by HTL activation, because no other RTK has been shown to be expressed in the mesoderm at the stage of gastrulation. Although the increased number of EVE-expressing cells detected in the dorsal mesoderm clusters following overexpression of Miple is eliminated in embryos lacking active HTL receptor, the possibility cannot be excluded that the lack of EVE-positive cells in the dorsal mesoderm might stem from the failure of the htl mutant mesoderm cells to reach the most dorsal locations (Toledano-Katchalski, 2007).

In vertebrates, midkine and pleiotrophin have been identified by phage display as potential high-affinity ligands for the human receptor tyrosine kinase ALK (Stoica, 2002). Although the possible role of Miple in ALK-dependent signaling cannot be excluded, ALK is not expressed in the early stages of mesoderm spreading, and does not overlap with the EVE-expressing clusters; thus, it is unlikely to affect the increased number of EVE-expressing cells. Receptor tyrosine phosphatase-zeta has been implicated as a putative pleiotrophin receptor (Milev, 1998). If a similar receptor exists in Drosophila, it might respond to Miple overexpression by altering MAPK levels (Toledano-Katchalski, 2007).

It is possible that the heparin-binding domain of Miple enhances the activity of the HTL ligands. In vertebrates, heparin-containing proteins act as co-ligands to FGFs by inducing their dimerization. Miple is a heparin-binding protein, because it binds specifically to a heparin column. The contribution of heparan sulfate proteoglycans to proper mesoderm spreading in Drosophila had been demonstrated by the requirement of two enzymes, Sugarless and Sulfateless, for this process. Moreover, a genetic interaction between mutations in each of these enzymes and the two FGF receptors HTL and Breathless (BTL) was demonstrated. Overexpression of Miple during mesoderm spreading might, on the one hand, compete with endogenous heparan sulfate proteoglycan for Thisbe and Pyramus binding, and thus could inhibit their ability to activate the HTL-dependent signaling. In contrast, Miple might also activate the HTL pathway by replacing the endogenous heparan sulfate proteoglycan that is normally involved in activation of the FGF8-like ligands. These dual activities might interfere with the normal dorsal-restricted MAPK activation in the mesoderm (Toledano-Katchalski, 2007).

In wild-type embryos, miple is downregulated by HOW(L) in the mesoderm; however, its mRNA expression is detected at later developmental stages, including in the ventral midline and in the brain (Englund, 2006). In midline glial cells, a second FGF receptor, Breathless, has been implicated in the promotion of cell migration at stages 12-13 of embryonic development. At this stage, Miple might contribute to the spatial and temporal control of Breathless activation. Such a scenario must be tested directly in miple mutant embryos (Toledano-Katchalski, 2007).

Although the mesoderm spreading phenotype of how germline clone embryos is not fully penetrant and is detected in only a few segments, the contribution of HOW activity is crucial because of the secondary effect that non-homogenous mesoderm spreading exhibits on the development of the heart and dorsal somatic mesoderm. HOW(L) appears to function in the mesoderm as a safety mechanism to prevent mis-expression of either maternally contributed genes or genes whose early transcriptional activation in the mesoderm might interfere with the normal development of the mesoderm. An example of similar repressive activity of HOW(L) is its activity in the reduction of string levels (Nabel-Rosen, 2005) in the gastrulating embryo to prevent premature cell division during mesoderm invagination (Toledano-Katchalski, 2007).

In summary, this study reveals the crucial function of the STAR family member HOW(L) in enabling proper mesoderm development via the repression of specific mRNAs provided either maternally, or expressed prematurely in a specific tissue. HOW(L) and its vertebrate homolog, QKI5, are expressed in wide range of tissues during early developmental stages, and might function in these tissues in a similar fashion to enable proper embryonic and tissue development (Toledano-Katchalski, 2007).

The pro-apoptotic activity of Drosophila Rbf1 involves dE2F2-dependent downregulation of diap1 and buffy mRNA

The retinoblastoma gene, rb, ensures at least its tumor suppressor function by inhibiting cell proliferation. Its role in apoptosis is more complex and less described than its role in cell cycle regulation. Rbf1, the Drosophila homolog of Rb, has been found to be pro-apoptotic in proliferative tissue. However, the way it induces apoptosis at the molecular level is still unknown. To decipher this mechanism, rbf1 expression was induced in wing proliferative tissue. It was found that Rbf1-induced apoptosis depends on dE2F2/dDP heterodimer, whereas dE2F1 transcriptional activity is not required. Furthermore, Rbf1 and dE2F2 downregulate two major anti-apoptotic genes in Drosophila: buffy, an anti-apoptotic member of Bcl-2 family and diap1, a gene encoding a caspase inhibitor. On the one hand, Rbf1/dE2F2 repress buffy at the transcriptional level, which contributes to cell death. On the other hand, Rbf1 and dE2F2 upregulate how expression. How is a RNA binding protein involved in diap1 mRNA degradation. By this way, Rbf1 downregulates diap1 at a post-transcriptional level. Moreover, the dREAM complex (see Rbf) has a part in these transcriptional regulations. Taken together, these data show that Rbf1, in cooperation with dE2F2 and some members of the dREAM complex, can downregulate the anti-apoptotic genes buffy and diap1, and thus promote cell death in a proliferative tissue (Clavier, 2014).

Targets of Activity

The selective sensitivity of cells to programmed cell death (PCD) depends on the positive and negative death-inducing signals that converge into the apoptotic pathway. In Drosophila, the midline glial (MG) cells undergo selective death during development. This study shows that the long isoform of the RNA-binding protein Held Out Wing (HOW(L)) is essential for enhancing the sensitivity of the MG cells to PCD. In how mutant embryos, the number of MG cells was elevated. This phenotype could be rescued by midline expression of the HOW(L) repressor isoform. In how mutant embryos, the levels of the caspase inhibitor of apoptosis, Diap1 were elevated, in parallel to reduction in the levels of activated caspase. Similarly, reducing the levels of HOW in S2 cells led to elevation of Diap1, whereas over expression of HOW(L) promoted reduction of Diap1 protein as well as mRNA levels. Importantly, deletion of the two HOW binding sites from diap1 3'UTR abrogated HOW-dependent repression of Diap1, suggesting that HOW represses diap1 by binding to its 3'UTR. These results suggest that HOW(L) enhances the sensitivity of MG cells to apoptotic signals by reducing the levels of diap1 in these cells in, demonstrating a novel mode of regulation of PCD at the mRNA level (Reuveny, 2009).

The sensitivity of cells to apoptotic signals depends on the balance between the pro-apoptic and anti-apoptotic signals expressed within a cell in a given developmental context. MG cells represent a unique system in which to study apoptosis because only a small subset of the cells (2-3 out of 6) are doomed to die, and the death must be executed in a relative short period of time (around 3 h), during the migration of the AMG pair towards the next segment. HOW functions to enhance the sensitivity of these cells to the pro apoptotic signals by reducing the levels of the anti-apoptotic protein, Diap1 (Reuveny, 2009).

Regulation through pro apoptotic signals, e.g. the activities of Reaper, Grim and Hid (RGH) proteins, or the anti-apoptotic signal, by influencing the activity of Diap1, enables cells to respond to a wide array of signaling pathways. The convergence of these signals in a single cell determines not only whether the cell will undergo PCD, but also the timing during development at which this process will occur. In case of MG cells, the timing is critical, as the cells die prior to their arrival to the commissure, and thus do not receive the survival signal through MAPK activation (Reuveny, 2009).

RGH proteins were shown to affect the levels of Diap1 at several regulatory stages. Reaper and Hid affect Diap1 levels via ubiquitination and proteosomal degradation. In addition, Morgue has been shown to promote Diap1 degradation whereas Reaper was shown to also inhibit translation of diap1. Despite the expression of Hid and Reaper in MG cells, only partial PCD is induced in these cells, possibly due to high levels of Diap1 (Reuveny, 2009).

Previous data has suggested that HOW(L) mediates developmental processes in other tissues (e.g., mesoderm, tendon cells), by a temporal reduction of the levels of key regulatory proteins. For example, in gastrulating embryos, HOW(L) reduces the mRNA levels of string/cdc25 to arrest cell division during mesoderm invagination, and at a later stage, HOW(L) reduces the levels of miple1 to allow mesoderm spreading. Similarly, this study shows that HOW contributes to the timing of MG cell apoptosis by reducing the levels of Diap1, thereby sensitizing these cells to pro-apoptotic signals. An effect of HOW on cell division through regulation of String is not favored by this study, since HOW is detected in the midline cells only at stage 12-13 at which the MG cells do not divide anymore (Reuveny, 2009).

Several lines of evidence support the idea that HOW(L) might affect MG cell apoptosis through its repression of Diap1 levels. First, it was shown by antibody staining that Diap1 levels are elevated in how mutants. Second, reducing HOW levels by introducing HOW-specific dsRNA or elevating HOW(L) levels in S2 cells leads to corresponding opposing effects on Diap1 protein expression, elevation of Diap1 when HOW is reduced and reduction of Diap1 when HOW(L) is elevated. The diap1 3' UTR contains two binding sites for HOW, and is capable of binding to HOW(L) in vitro. Nevertheless, a corresponding elevation of diap1 mRNA could not be detected in the S2 cells in which HOW was knocked down by dsRNA, possibly due to a continuous positive transcriptional input of diap1 in these cells. However, a reduction of diap1 mRNA and protein levels was induced, following over expression of HOW(L) in S2 cells and in embryos. Also, whether the splicing pattern of diap1 was altered in S2 cells depleted of HOW was examined, since HOW was demonstrated to mediate alternative splicing in other tissues. To this end, an RT-PCR was performed with primers specific for each of the three diap1 splice variants; however, no change in the pattern of diap1 splicing was observed (Reuveny, 2009).

Thus, HOW(L) might affect Diap1 protein levels by repressing both its mRNA levels as well as its translation. Alternatively it could affect Diap1 indirectly by influencing the levels of an upstream regulator of Diap1. The results support a direct effect of HOW through its association with the HOW-binding sites in diap1 3' UTR, since deletion of these sites abrogated the reduction of Diap1 detected in the presence of HOW(L) (Reuveny, 2009).

Gld-1 the C. elegans orthologue of HOW affects both translation and stability of its target mRNAs, apparently by affecting the length of the polyA tail of the target mRNA. HOW(L) might act in a similar fashion on diap1 mRNA (Reuveny, 2009).

Whereas HOW(L) does not induce apoptosis in other tissues, where it is highly expressed (e.g. mesoderm, tendon cells etc.), it was shown to have pro apoptotic effects in MG cells and in the adult fly eye. It is suspected that in these tissues, a delicate balance between the levels of the pro apoptotic and anti apoptotic proteins is maintained, so that the cells become highly sensitive to Diap1 levels, and thus are responsive to reduced or elevated levels of HOW(L) (Reuveny, 2009).

Interestingly, one isoform of the mammalian orthologue of HOW, Quaking7 (QKI-7), has been shown to induce apoptosis of fibroblasts and primary rat oligodendrocytes. The molecular mechanism of QKI-7-induced apoptotic activity has yet to be elucidated, but the unique C' terminal tail of QKI-7 appears to be necessary for this apoptotic activity. In contrast, C. elegans GLD-1 was shown to repress the levels of cep1 an orthologue of mammalian P53. In that system, GLD-1 exhibits an anti apoptotic effect. It appears therefore, that STAR proteins are not dedicated to a defined direction of apoptotic regulation. Rather, their basic activity is to elevate or reduce the levels of critical components in the process to enable the execution of PCD or to allow other developmental process to occur (Reuveny, 2009).

Previous studies demonstrated that how transcription is induced in response to the activation of the Ecdyson (Ecd) pathway however, the biological significance of this induction was not clear. During larval stages, a high titer of Ecd acts through the ecdyson receptor EcR/Ultraspiracle nuclear receptor heterodimer to signal puparium formation and destruction of several larval tissues including the midgut and salivary glands. The Ecd pathway triggers a transcriptional cascade that culminates in rpr and hid induction to initiate tissue destruction. Interestingly, the Ecd pathway induces parallel repression of diap1 via the activity of the CREB binding protein, CBP. CBP is both necessary and sufficient to down-regulate Diap1, providing the cells with the competence to die. Whereas the contribution of CBP to MG cell apoptosis has yet to be elucidated, it is possible that in this system, in parallel to the induction of CBP transcription, the Ecd pathway triggers HOW(L) transcription to enhance Diap1 destruction, possibly due to a need to induce rapid death of the MG cells (Reuveny, 2009).

To address whether HOW(L) is sufficient to rescue the excess in MG cells in disembodied (dib) mutant embryos, defective in ecdysone biosynthetic, HOW(L) was overexpressed in dib mutant embryos that carried the MG-specific enhancer trap, AA142, using the sim-gal driver. Embryos over expressing HOW(L) in dib mutant embryos still maintained a high number of MG cells, suggesting that HOW(L) is not sufficient to rescue the dib mutant phenotype. Importantly, the levels of Diap1 in MG cells in these embryos were significantly reduced (Reuveny, 2009).

The inability of HOW(L) to reduce the number of MG cells following its over expression in the dib mutant embryos might be explained by the involvement of the Ecd pathway not only in PCD but also in repression of MG cell division in an earlier developmental stage. Also, since the Ecdysone pathway positively regulates Hid, it is possible that the MG cells did not contain enough pro-apoptotic signals to induce PCD, and therefore it is not surprising that HOW(L) did not provide rescue of the MG cell number (Reuveny, 2009).

In summary, this study have identified the KH-domain RNA-binding protein, HOW, as a novel regulator of PCD in MG cells, likely acting as a regulator of Diap1 translation and/or stability. It is proposed that HOW provides the MG cells with enhanced sensitivity to the pro apoptotic effects of Hid and Reaper, triggering the rapid apoptosis of MG cells during their migration (Reuveny, 2009).

The regulation of glial-specific splicing of Neurexin IV requires HOW and Cdk12 activity

The differentiation of the blood-brain barrier (BBB) is an essential process in the development of a complex nervous system and depends on alternative splicing. In the fly BBB, glial cells establish intensive septate junctions that require the cell-adhesion molecule Neurexin IV. Alternative splicing generates two different Neurexin IV isoforms: Neurexin IVexon3, which is found in cells that form septate junctions, and Neurexin IVexon4, which is found in neurons that form no septate junctions. This study shows that the formation of the BBB depends on the RNA-binding protein HOW (Held out wings), which triggers glial specific splicing of Neurexin IVexon3. Using a set of splice reporters, it was shown that one HOW-binding site is needed to include one of the two mutually exclusive exons 3 and 4, whereas binding at the three further motifs is needed to exclude exon 4. The differential splicing is controlled by nuclear access of HOW and can be induced in neurons following expression of nuclear HOW. Using a novel in vivo two-color splicing detector, a screened was carried out for genes required for full HOW activity. This approach identified Cyclin-dependent kinase 12 (Cdk12) and the splicesosomal component Prp40 as major determinants in regulating HOW-dependent splicing of Neurexin IV. Thus, in addition to the control of nuclear localization of HOW, the phosphorylation of the C-terminal domain of the RNA polymerase II by Cdk12 provides an elegant mechanism in regulating timed splicing of newly synthesized mRNA molecules (Rodrigues, 2012).

Cdk12 protein is a nuclear localized kinase that phosphorylates the C-terminal domain (CTD) of the RNA polymerase II during transcriptional elongation (Bartkowiak, 2010). The phosphorylated CTD is bound by Prp40, a subunit of the U1 snRNP. Prp40, in turn, has been shown to interact with the HOW-binding protein Crn/Clf1. Thus, Cdk12 is in a position to facilitate splicing of pre-mRNAs that have bound the HOW protein (Rodrigues, 2012).

Differential splicing is a key element in generating the amazing complexity of higher nervous systems. Through relatively few regulatory elements, a single gene can generate several different isoforms with potential distinct cellular functions. In Drosophila, differential splicing is required for the correct glial development. This study has dissected the role of the STAR-family member HOW in controlling such a differential splicing event at the Nrx-IV locus, which is pivotal for the generation of the BBB (Rodrigues, 2012).

Nrx-IV exons 3 and 4 are spliced in a mutually exclusive manner. They share DNA sequence identity of 60% and encode related Discoidin domains, which provide distinct adhesive properties. Within glial cells, expression of Nrx-IVexon3 predominates and participates in the formation of septate junctions. Interestingly, the binding partner of Nrx-IV at the Drosophila septate junctions, Neuroglian, or the Caspr-binding partner at the septate-like junctions in vertebrates, Neurofascin, are also subject to cell-type specific, differential splicing (Rodrigues, 2012).

Differential splicing appears to be of more general relevance during the formation of septate junctions. The fly homologue of the membrane-skeleton protein 4.1, Coracle, binds to Nrx-IV and mediates the linkage of the septate junctions to the cytoskeleton. Differential splicing of coracle generates at least four different splice variants that encode four distinct proteins. RT-PCR experiments indicate that the Coracle-PB isoform is generated in a HOW-dependent manner (Rodrigues, 2012).

STAR proteins, like HOW, bind sequence motifs in the pre-mRNA of their targets. Following site-specific mutation of all HOW response elements (HREs), it was shown that HRE1 may be needed for general exon definition. The mutation of this sequence motif leads to increased exon skipping of both exon 3 and exon 4, suggesting a crucial role for HRE1 in general splicing, possibly affecting the branch point of this intron. The HRE2, HRE3 and HRE4 elements influence mutually exclusive splicing. Upon mutation of these motifs, both exons are left in the mRNA more frequently, which suggests their function in exon selection. Such an effect was not observed in neurons. Thus, these HREs seem to play a role in exon selection (Rodrigues, 2012).

The HOW isoforms share an identical KH RNA-binding domain. HOW(S) predominantly localizes to the cytoplasm and HOW(L) is found mostly in the nucleus of glial cells. This study showed that nuclear HOW is sufficient for the induction of glial-specific splicing in neurons. Interestingly, both HOW isoforms can partially rescue the how mutant phenotype. HOW(S) appears to have higher rescuing abilities. As both transgenes are inserted in the same chromosomal landing site, resulting in identical expression levels, it is assumed that HOW(S) must be efficiently transported into the nucleus to promote Nrx-IV splicing. Because, following overexpression of the HOW(S), most of the protein stays in the cytoplasm, the shuttle mechanism(s) directing HOW(S) into the nucleus must be very tightly regulated. Possibly, HOW(S) has better rescuing abilities as HOW(S), but not HOW(L), can facilitate the nuclear import of the splice factor Crn (Rodrigues, 2012).

STAR family proteins are phosphorylated on several residues. In the past, it has been established that the HOW homolog Sam68 is phosphorylated by MAPK the regulation of which is controlled by Raf. Indeed, expression of a dominant-negative Raf protein in glia shifted the splicing pattern towards the neuronal form, suggesting a role for receptor tyrosine kinase signaling for glial differentiation as it has been demonstrated at several other instances (Rodrigues, 2012).

In addition, it is noted that silencing of Cdk12 resulted in a shift of the splicing pattern towards the neuronal form. Cdk12 is a broadly expressed serine/threonine kinase that also contains stretches of arginine- and serine-rich sequences (SR domains) known to be present in RNA-processing proteins, which regulate splicing, nuclear export and stability of the mRNA. Drosophila Cdk12 is associated with the C-terminal domain (CTD) of the RNA polymerase II (RNAPII) and phosphorylates Ser2 (Bartkowiak, 2010). The CTD of RNAPII acts as an assembly platform that controls transcription and pre-mRNA processing. Phosphorylated CTD in turn is recognized by Prp40, which belongs to the U1 snRNP. Moreover, a direct interaction between PrP40 and Crocked neck like factor 1 (Clf1), which binds HOW, has been demonstrated. Thus, phosphorylation of CTD by Cdk12 (Bartkowiak, 2010) recruits the assembly of the spliceosome at specific pre-mRNA targets defined by binding of HOW. In line with this model, it is noted that silencing of Prp40 also alters Nrx-IV splicing (Rodrigues, 2012).

In Drosophila, Cdk12 associates with Cyclin K (Bartkowiak, 2010), which is required for its catalytic activity. The activity of cyclins can be regulated by RTK signaling and thus might present a link that connects the Raf/MAPK pathway with a direct control of splicing activity. Additionally, CTD phosphorylation could be linked to MAPK activity in former studies. Cdk12 is expressed in the nucleus of almost all cells. To further decipher the role of Cdk12 during splicing, a loss-of-function allele was used. Homozygous mutant animals are lethal at the beginning of larval development. However, these mutants show no splicing defects, most probably owing to strong maternal contributions (Rodrigues, 2012).

The formation of the BBB implies the maturation of septate junctions only in fully differentiated subperineurial glial cells. Thus, the timing of splicing of pre-mRNAs encoding septate junction proteins is crucial and most likely regulated by two independent signaling cascades. It is proposed that the mRNA-binding protein HOW integrates these signaling events and is key in determining cellular differentiation (Rodrigues, 2012).

The Drosophila RNA-binding protein HOW controls the stability of dgrasp mRNA in the follicular epithelium

Post-transcriptional regulation of RNA stability and localization underlies a wide array of developmental processes, such as axon guidance and epithelial morphogenesis. In Drosophila, ectopic expression of the classically Golgi peripheral protein dGRASP at the plasma membrane is achieved through its mRNA targeting at key developmental time-points, in a process critical to follicular epithelium integrity. However, the trans-acting factors that tightly regulate the spatio-temporal dynamics of dgrasp are unknown. Using an in silico approach, two putative HOW Response Elements (HRE1 and HRE2) were identified within the dgrasp open reading frame for binding to Held Out Wings (HOW), a member of the Signal Transduction and Activation of RNA family of RNA-binding proteins. Using RNA immunoprecipitations, this was confirmed by showing that the short cytoplasmic isoform of HOW binds directly to dgrasp HRE1. Furthermore, HOW loss of function in vivo leads to a significant decrease in dgrasp mRNA levels. HRE1 protects dgrasp mRNA from cytoplasmic degradation, but does not mediate its targeting. It is proposed that this binding event promotes the formation of ribonucleoprotein particles that ensure dgrasp stability during transport to the basal plasma membrane, thus enabling the local translation of dgrasp for its roles at non-Golgi locations (Giuliani, 2013).

The functional HRE is situated in the dgrasp ORF, and not in the 3'UTR as classically reported for QKI/GLD-1 targets. Interestingly, the C. elegans homologue of dgrasp mRNA also displays two predicted HREs in its ORF, suggesting that this feature is conserved. RBPs binding mRNAs in the ORF (including members of the STAR family) are an emerging trend in RNA metabolism with roles in translational repression, transport, as well as stabilization. The study of these new targets will open new avenues in the understanding of how RNA metabolism is regulated. In this respect, a crucial step is to determine whether a functional biological bias exists between the bindings of RBPs in the ORF versus the UTRs of a transcript (Giuliani, 2013).

Protein Interactions

The splicing factor crooked neck associates with the RNA-binding protein HOW to control glial cell maturation in Drosophila

In both vertebrates and invertebrates, glial cells wrap axonal processes to ensure electrical conductance. Crooked neck (Crn), the Drosophila homolog of the yeast Clf1p splicing factor, directs peripheral glial cell maturation. crooked neck is expressed and required in glial cells to control migration and axonal wrapping. Within the cytoplasm, Crn interacts with the RNA-binding protein HOW and then translocates to the nucleus where the Crn/HOW complex controls glial differentiation by facilitating splicing of specific target genes. By using a GFP-exon trap approach, some of the in vivo target genes were identified that encode proteins localized in autocellular septate junctions. Thus, glial cell differentiation is controlled by a cytoplasmic assembly of splicing components, which upon translocation to the nucleus promote the splicing of genes involved in the assembly of cellular junctions (Edenfeld, 2006).

Most glial cells of the Drosophila PNS are born in the CNS and migrate toward their final destination. Subsequently, the cell body follows to then initiate wrapping of the axons. crn and how are both required for the initiation of axonal wrapping. The proteins encoded by crn and how are involved in the regulation of splicing of components of the septate junctions that are required for glial cell differentiation. Crn and HOW(S) interact in the cytosol to control their nuclear import, providing a simple mechanism to couple glial and neuronal cell differentiation (Edenfeld, 2006).

crn encodes an unusual TPR-containing protein whose function is essential for embryonic development. The Crn protein is found in the cytosol and in nuclear “speckles” (Raisin-Tani, 2002). Previous genetic and biochemical evidence has already suggested that Crn and its homologs participate in the assembly and the control of the splicing machinery. A mutation of the yeast crooked neck ortholog results in the accumulation of unspliced pre-mRNAs and, furthermore, Crn-like proteins are needed for pre-mRNA splicing in vitro. Crn is found in two functional complexes with and without snRNA and via its N-terminal TRPs helps to assemble the intact spliceosome (Wang, 2003). Within the spliceosome, the Crn homolog assists in the initial spliceosome assembly and also binds the phospho-CTD of the RNA polymerase II (Gasch, 2005). In Drosophila crn mutants, changes were observed in the splicing pattern suggesting that Crn modulates splicing preferences during alternative splicing (Edenfeld, 2006).

Alternative splicing employs differential use of 5' or 3' splice sites and has evolved as an efficient way to achieve a functional diversification and regulation of gene products. The basic splicing mechanism first requires the correct choice of 5' and 3' splice junctions and subsequently the assembly of the spliceosome. While Crn can facilitate spliceosome assembly, it does not directly participate in the selection of specific splice junctions, since the Crn protein is not able to bind to RNA. However, in Drosophila, Crn does regulate alternative splicing of few specific target genes, implying the existence of interaction partners that direct the Crn protein to these target RNAs (Burnette, 1999; Park, 2004; Edenfeld, 2006 and references therein).

This study has identified the HOW(S) protein as such an interaction partner that is likely able to recruit Crn to specific splicing targets. The HOW proteins contain an hnRNP K homology (KH) motif and exhibit specific RNA-binding activities. The KH motif is found in the GSG domain (GRP33, Sam68, GLD) shared by the Signal Transduction and Activation of RNA (STAR) family of proteins. The how locus is genetically complex: it encodes two antagonizing splice variants, HOW(S) and HOW(L), the functions of which were thoroughly analyzed in tendon cells. This study shows that HOW(L) is involved in the instability of stripe mRNAs, whereas HOW(S) is involved in mRNA stability as well as in the control of stripe A splicing (Volohonsky, 2007). Since HOW(S) associates with Crn, a direct influence on splicing can be anticipated (Edenfeld, 2006).

The proposed cytoplasmic-nuclear shuttling of a Crn/HOW(S) complex furthermore allows the linking of extracellular signals to a direct control of splicing. Concerning glial cell differentiation, this suggests that wrapping of axonal fascicles is not only dependent on a transcriptional control. It is rather likely that neuronal signals help to efficiently couple glial and neuronal differentiation by directly influencing the splicing pattern. The nature of such a signal is still elusive, however. As described for the HOW-related protein Sam68, phosphorylation may be important to control the interaction of Crn and HOW in the cytoplasm and thus the transport of the complex into the nucleus. In this respect, it is interesting to note that the development of oenocytes, which is impaired in both crn and how mutants, requires EGF-receptor signaling and that neuronal EGF-receptor signaling has been shown to regulate glial expression of neuroglian (Edenfeld, 2006).

In agreement with such a model is the finding that the how mutant phenotype resembles the phenotype caused by the loss of crn. Furthermore, Crn is able to bind only cytosolic HOW(S). If HOW(L) expression is forced to the cytosol, it can also bind to Crn, confirming that the interaction of Crn and HOW occurs in the cytosol. The assembly of the Crn/HOW complex is crucial to precisely regulate the nuclear concentration of these splice factors, which in turn is relevant for alternative splicing. Within the nucleus, HOW(S) binds to a consensus sequence with a length of only five nucleotides, making the quest for specific target genes difficult. To nevertheless get insight into this important functional aspect, a collection of strains was utilized in which endogenous genes were tagged by the insertion of a GFP-exon. In this study two components of the septate junctions were identified that form important autocellular junctions needed to stabilize glial cell morphology as candidate targets for Crn. Glial septate junctions are morphologically established by the end of embryogenesis. In line with the notion that neurexinIV is a target gene of crn, no septate junctions were detected in crn mutant glial cells. Furthermore, dye-penetration experiments show that the blood-brain barrier, which crucially depends on the presence of septate junctions, is not established in crn mutants (Edenfeld, 2006).

In addition to regulating splicing, Crn and HOW proteins may also have additional functions. For example, members of the hnRNP-A/B family of RNA-binding proteins are able to regulate alternative splicing of the Drosophila P element transposase and the Ubx gene. However, the function of the hnRNP-A/B family member Hrp48 is not restricted to the control of RNA splicing since it is also involved in the control of oskar mRNA localization in the Drosophila oocyte. Such a dual specificity of the Hrp48 RNA-binding protein in regulating RNA splicing and RNA transport has also been suggested for the Crn-binding partner HOW. In addition, it was recently demonstrated that the yeast Crn homolog also affects DNA replication, and first phenotypic analyses of the Drosophila crn mutant led to the proposal that Crn might play a role in regulation of cell divisions (K. Zhang, 1991). However, no abnormal cell number was observed for the peripheral glial cells, suggesting that at least during glial development crn has no function during the cell cycle (Edenfeld, 2006).

The data lead to a model underlying glial cell differentiation that may not only be applicable for Drosophila. The vertebrate homolog of how is the quaking gene, which is also required for glial differentiation. quaking viable mutants initially develop normally but then show tremors due to severe myelination defects. The mutant phenotype is caused by a deletion in the promoter region of the quaking gene that encodes several alternatively spliced mRNAs. The quaking viable deletion abrogates the expression of QKI-6 and QKI-7 in myelinating cells of the brain. The complete loss of quaking transcripts results in early lethality. quaking and how mutants not only share a defect in axonal wrapping. Moreover, the corresponding gene products appear to have different functions in the nucleus and the cytosol. Whereas QKI-5 is strictly nuclear, QKI-6 and QKI-7 are able to shuttle between the cytosol and the nucleus as it has been observed for the HOW(S) protein. The position of putative QKI binding sites close to tissue-regulated exons was found to be conserved in mice and is similar to what was found for the putative HOW binding sites in neurexinIV. Thus, although invertebrates and vertebrates have long been thought to follow very different routes toward glial differentiation, the underlying molecular control of glial wrapping may be conserved (Edenfeld, 2006).

Phosphorylation of the Drosophila melanogaster RNA-binding protein HOW by MAPK/ERK enhances its dimerization and activity

Drosophila melanogaster Held Out Wings (HOW) is a conserved RNA-binding protein (RBP) belonging to the STAR family, whose closest mammalian ortholog Quaking (QKI) has been implicated in embryonic development and nervous system myelination. The HOW RBP modulates a variety of developmental processes by controlling mRNA levels and the splicing profile of multiple key regulatory genes; however, mechanisms regulating its activity in tissues have yet to be elucidated. This study links receptor tyrosine kinase (RTK) signaling to the regulation of QKI subfamily of STAR proteins, by showing that HOW undergoes phosphorylation by MAPK/ERK. Importantly, that this modification facilitates HOW dimerization and potentiates its ability to bind RNA and regulate its levels. Employing an antibody that specifically recognizes phosphorylated HOW, this study shows that HOW is phosphorylated in embryonic muscles and heart cardioblasts in vivo, thus documenting for the first time Serine/Threonine (Ser/Thr) phosphorylation of a STAR protein in the context of an intact organism. The sallimus/D-titin (sls) gene was identified as a novel muscle target of HOW-mediated negative regulation and further show that this regulation is phosphorylation-dependent, underscoring the physiological relevance of this modification. Importantly, it was demonstrated that HOW Thr phosphorylation is reduced following muscle-specific knock down of Drosophila MAPK rolled and that, correspondingly, Sls is elevated in these muscles, similarly to the HOW RNAi effect. Taken together, these results provide a coherent mechanism of differential HOW activation; MAPK/ERK-dependent phosphorylation of HOW promotes the formation of HOW dimers and thus enhances its activity in controlling mRNA levels of key muscle-specific genes. Hence, these findings bridge between MAPK/ERK signaling and RNA regulation in developing muscles (Nir, 2012).


DEVELOPMENTAL BIOLOGY

Embryonic

In spite of the strong maternal expression of the 3.6 kb transcript, no protein is detected before the onset of gastrulation, suggesting that the maternally supplied mRNA is either not translated or that the translated protein is highly unstable (Zaffran, 1997).

how mRNA is first detected in mesodermal precursors on the ventral side of the embryo at the onset of gastrulation. how transcription continues to be detected in presumptive mesoderm cells at the germ band extension stage. As germ band retraction occurs, how transcription is detected in the cells destined to form somatic and visceral muscle cells. Expression is also detected in the pharyngeal muscles at the anterior end of the embryo. how transcription is detected in cardiac precursors and muscle attachment cells of the epidermis as dorsal closure occurs. Late during embyogenesis, how transcription is restricted to the heart and muscle attachment sites of the epidermis (Baehrecke, 1997).

When the different mesodermal lineages segregate, the expression of How becomes restricted to the myogenic lineage, including the cardioblasts and probably all the myoblasts. Antibodies directed against the protein demonstrate that How is localized to the nucleus (Zaffran, 1997).

Genetic control of cell morphogenesis during Drosophila melanogaster cardiac tube formation

Tubulogenesis is an essential component of organ development, yet the underlying cellular mechanisms are poorly understood. This study analyzed the formation of the Drosophila cardiac lumen that arises from the migration and subsequent coalescence of bilateral rows of cardioblasts. This study of cell behavior using three-dimensional and time-lapse imaging and the distribution of cell polarity markers reveals a new mechanism of tubulogenesis in which repulsion of prepatterned luminal domains with basal membrane properties and cell shape remodeling constitute the main driving forces. Furthermore, a genetic pathway is identified in which roundabout, slit, held out wings, and dystroglycan control cardiac lumen formation by establishing nonadherent luminal membranes and regulating cell shape changes. From these data a model is proposed for Drosophila cardiac lumen formation, which differs, both at a cellular and molecular level, from current models of epithelial tubulogenesis. It is suggested that this new example of tube formation may be helpful in studying vertebrate heart tube formation and primary vasculogenesis (Medioni, 2008).

The analysis provided here establishes the cellular basis of lumen formation of the Drosophila cardiac tube. The lumen of the tube is formed from the migration of two bilateral rows of polarized cardioblasts (CBs), which join at the dorsal midline. One main result of this study is the characterization of two types of cell membrane domains directly involved in lumen formation, the luminal domains (L domains) and adherent domains (J domains). Adherens junctions that are responsible for sealing the tube originate from the J domain, whereas the membrane walls of the lumen originate from the L domain (Medioni, 2008).

Remarkably, the L domain displays characteristics of basal membranes, revealed by expression of molecular markers normally associated with a basal membrane. Furthermore, specification of the L and J domains takes place very early in the tubulogenesis process, significantly before coalescence of the bilateral rows of CBs at the dorsal midline. Finally, during CB migration, membrane domains undergo remodeling, concomitant with profound cell shape changes. These two cellular processes appear to be closely connected and are probably regulated by the cellular environment of the CBs composed by the overlying dorsal ectoderm and the amnioserosa cells. These interactions will be investigated in a future work (Medioni, 2008).

The mechanism of Drosophila cardiac lumen formation reported in this study is thus notably different from the previously described mechanisms of epithelial tubulogenesis. In epithelial tubulogenesis, after receiving a polarization signal that sets apicobasal polarity, the cells or group of cells establish a basal surface and generate vesicles carrying apical membrane proteins. The vesicles are targeted to the prospective apical region, where they fuse with the existing membrane or with each other to form a lumen. Finally, continued vesicle fusion and apical secretion expand the lumen (Medioni, 2008).

In contrast, constriction of the leading edge domain during cardioblast (CB) migration, precise control of cell shape changes, and delimitation of specific membrane domains appear to be the driving forces of Drosophila cardiac lumen formation. Cells forming the dorsal vessel have the features of migrating cells. In contrast to epithelial tubulogenesis, which involves apical membrane domains, the apex of polarized CBs constricts, forms adherens junctions, and consequently does not constitute the L domain. Instead, the luminal membrane domain possesses basal membrane characteristics, as is also the case in endothelial cells. Moreover, the size of the cardiac lumen is determined by the isotropic growth of CBs, and not, as in other models, by anisotropic extension of the L domain involving apical membrane vesicles.

Finally, the genetic control of the process involves gene products of slit, robo, how, and dg, which are not known regulators of lumen formation in epithelial tubes (Medioni, 2008).

This study leads to the identification of a genetic pathway, including slit, robo, how, and dg, controlling membrane domain specification and dynamics during cardiac lumen formation. Within this pathway, Slit appears to play a central role and a previously unrecognized function in cell morphogenesis (Medioni, 2008).

Several studies have shown that Slit-Robo function is essential for cardiac tube formation by controlling the proper migration, cohesion, and alignment of the two rows of CBs. The results reported in this study show that Slit is also involved in the correct specification of the L domain and its distinct features with respect to the adjacent J domains. Activation of Slit-Robo signaling determines the respective size of these two types of domains (Medioni, 2008).

The data suggest that activation of this pathway inhibits the formation of adherens junctions. This possibility is supported by recent findings in chick retina cells, where activation of the Slit-Robo pathway leads to the inactivation of β-catenin (Arm in Drosophila), resulting in the dissociation of N-cadherin from the junctional complex and preventing the formation of adherens junctions. Consistent with these observations, DE-Cad (Shg) is expressed in the J domains of CBs and is required for cardiac tube morphogenesis. Moreover, slit and shg show genetic interaction in cardiac tube morphogenesis. In the absence of slit function, the size of the L domain is strongly reduced, suggesting that Slit-Robo signaling prevents the formation of Arm/DE-Cad-mediated adherens junctions in the L domain (Medioni, 2008). How encodes an RNA-binding protein involved in mRNA metabolism, and given its exclusive nuclear localization at this stage of development, How may regulate slit splicing. In the absence of the How protein, the gene splicing could be affected, producing a Slit protein unable to correctly localize at the L domain. This hypothesis is consistent with the fact that expression of wild-type Slit in CBs can suppress the effect of how18 mutation on Slit localization and lumen formation. How has also recently been shown to regulate the splicing of neuronal membrane proteins such as neurexin. Moreover, How is expressed in the midline glia with Slit and Dg, suggesting that interaction among these three genes is part of a general mechanism by which junctions and lumen formation are controlled (Medioni, 2008).

A model is preposed for the genetic control of lumen formation in the cardiac tube. According to this model, How could directly regulate Slit by controlling its splicing and targeting the luminal compartment. Consequently, Slit binds to Robo activating the signaling pathway, which in turn inhibits Arm/DE-Cad-mediated adherens junction formation in the luminal compartment, leading then to the specification of distinct J and L domains. Parallel to this, activation of Slit-Robo signaling modulates the actin cytoskeleton and triggers CB cell shape remodeling required for lumen formation and growth. As How is able to act on many targets, it could also directly control the actin cytoskeleton by targeting an actin-binding molecule. Concerning Dg, it was shown that dg and slit genetically interact; however, overexpression of Slit does not rescue the lumen phenotype observed in dg mutants, contrasting with how mutations. Thus, it is proposes that Dg could regulate Slit localization at the L domain by its function in the specification and differentiation of the L domain, and therefore acts parallel to slit for lumen formation, behaving, for example, as a coreceptor of Robo. In addition, Dg could control actin cytoskeleton dynamics via its interaction with Dystrophin (Medioni, 2008).

The data clearly show that cardiac tube formation in Drosophila differs substantially from all other described mechanisms of tubulogenesis. Is this mechanism of tubulogenesis unique or is it shared with other organs and/or other organisms? Primary vasculogenesis in vertebrates leads to the formation of large median vessels, the dorsal aorta and the cardinal vein. These vessels arise from migrating mesenchymal cells of the lateral mesoderm, termed angioblasts, that are organized in bilateral groups of cells. Angioblasts migrate toward the midline as a cohort of cells, coalesce, and form a lumen. At this stage, as in flies, cells around the lumen show a crescentlike shape and an extracellular matrix is deposited at the internal face of luminal membranes. Similar cellular events are also observed during the formation of the primitive cardiac tube in vertebrates, suggesting that a common mechanism of tubulogenesis might exist for all tubes that arise from the coalescence of migrating bilateral mesenchymal cells (Medioni, 2008).

The Drosophila cardiac tube, or dorsal vessel, shares many similarities with the cardiovascular system of vertebrates. A significant fraction of genes expressed in the Drosophila cardiac tube are also annotated to be expressed in vertebrate blood vessels, suggesting that vasculogenesis and dorsal vessel morphogenesis might share common genetic regulators (Medioni, 2008).

Finally, components of the genetic pathway controlling cardiac lumen formation that are described in this study have potentially similar functions in vertebrates. It has been shown that numerous proteins involved in axon guidance are expressed in vertebrate blood vessels. In particular, the Slit-Robo signaling pathway has been involved in promoting tumor vascularization, hSlit2 being expressed in tumor cells and hRobo1 in endothelial cells. Moreover, mSlit3 has been implicated in mammalian cardiogenesis, and Quaking, the mouse homologue of How, is required for vasculogenesis and expressed in the developing heart (Medioni, 2008).

In conclusion, analysis of CB morphogenesis during development of the Drosophila cardiovascular system provides evidence for a new model of biological tube formation. It is proposed that this mechanism might also be used for the formation of the large median vessels and primitive heart tube in vertebrates (Medioni, 2008).

Larval

how transcription increases at metamorphosis. Similar to the pattern of expression during embryogenesis, how transcription is detected in muscle and muscle attachment cells during the onset of metamorphosis. how is detected in adult muscle cell precursors that are attached to the wing imaginal disc (Baehrecke, 1997).


EFFECTS OF MUTATION

Animals lacking how function die late during embryogenesis, possessing defects that seem to occur sometime between myoblast fusion and muscle cell attachment. A lethal mutant dies with the most posterior region of the cuticle arrested above the dorsal surface, presumably cause by failure to complete germ band retraction. howe44 mutants have normal differentiated and fused myotubules, but with variable expressivity the myotubes of some embryos appear to migrate but are disorganized and don't possess the proper pattern of attachment while the myotubules of other embryos show no signs of migration and actually appear to have fewer myoblasts. Partial loss-of-function results in lethality during the metamorphosis from a larva to an adult. Most howr17 homozygotes die during metamorphosis with their heads stuck inside their thoraxes. A small number of howr17 homozygotes escape as adults that do not fold their wings properly and, thus, possess the phenotype for which this gene is named. These flies also have blisters on their wings (Baehrecke, 1997).

Hypomorphic mutant alleles of how produce adult animals that have lost the ability to fly; they keep their wings on the horizontal, at a 90∞ angle to the body axis (Zaffran, 1997).


EVOLUTIONARY HOMOLOGS

GLD-1, a KH domain RNA-binding protein of C. elegans

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).

Germ cells, the cells that give rise to sperm and egg, maintain the potential to recreate all cell types in a new individual. This wide developmental potential, or totipotency, is manifested in unusual tumors called teratomas, in which germ cells undergo somatic differentiation. Although recent studies have implicated RNA regulation, the mechanism that normally prevents the loss of germ cell identity remains unexplained. In C. elegans, a teratoma is induced in the absence of the conserved RNA-binding protein GLD-1. Here, this study demonstrates that GLD-1 represses translation of CYE-1/cyclin E during meiotic prophase, which prevents germ cells from re-entering mitosis and inducing embryonic-like transcription. A mechanism is described that prevents precocious mitosis in germ cells undergoing meiosis, it is proposed that this mechanism maintains germ cell identity by delaying the onset of embryonic gene activation until after fertilization, and a paradigm is provided for the possible origin of human teratomas (Biedermann, 2009).

Specialized ribonucleoprotein organelles collectively known as germ granules are found in the germline cytoplasm from worms to humans. In Drosophila, germ granules have been implicated in germline determination. C. elegans germ granules, known as P granules, do not appear to be required for primordial germ cell (PGC) determination, but their components are still needed for fertility. One potential role for P granules is to maintain germline fate and totipotency. This is suggested by the loss of P granules from germ cells that transform into somatic cell types, e.g., in germlines lacking MEX-3 and GLD-1 (Drosophila homolog: Held out wings) or upon neuronal induction by CHE-1 (Drosophila homolog: Glass). However, it has not been established whether loss of P granules is the cause or effect of cell fate transformation. To test cause and effect, P granules were severly compromised by simultaneously knocking down factors that nucleate granule formation (PGL-1 and PGL-3) and promote their perinuclear localization [GLH-1 (see Drosophila Vasa) and GLH-4] and an investigation was carried out to see whether this causes germ cells to lose totipotency and initiate somatic reprogramming. It was found that compromising P granules causes germ cells to express neuronal and muscle markers and send out neurite-like projections, suggesting that P granules maintain totipotency and germline identity by antagonizing somatic fate (Updike, 2014).

Fish and frog quaking homologs

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).

Cloning and characterization of murine quaking family members

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 qkIkt4 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 (qkv) is a well known dysmyelination mutation. Recently, the genetic lesion of qkv 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 qkv/qkv brain during early myelination. In addition, markedly reduced MBP mRNAs are detected in the qkv/qkv 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 qkv 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 (qkv) 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 qkv dysmyelination remains unclear. Distinct molecular mechanisms underlie the reduction of MBP and the 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP) in qkv dysmyelination. MBP transcripts bind QKI strongly and are markedly reduced in the qkv/qkv 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 qkv/qkv brain. Since CNP transcripts are predominantly associated with translating polyribosomes, diminished CNP expression in qkv dysmyelination is unlikely to be due to translational failures, but more likely results from accelerated protein degradation (Zhang, 2001).

Sam68, a KH domain and RNA binding protein that is a substrate of Src tyrosine kinases

Sam68 is a member of a growing family of RNA-binding proteins that contains an extended K homology (KH) domain embedded in a larger domain, called the GSG (GRP33, Sam68, GLD1) domain. To identify GSG domain family members, data bases were searched for expressed sequence tags encoding related portions of the Sam68 KH domain. Two novel Drosophila KH domain proteins, which have been termed KEP1 (KH encompassing protein) and SAM, were identified. SAM bears sequence identity with mammalian Sam68 and may be the Drosophila Sam68 homolog. SAM, KEP1, and the recently identified Drosophila Who/How are RNA-binding proteins that are able to self-associate into homomultimers. The GSG domain of KEP1 and SAM is necessary to mediate the RNA binding and self-association. To elucidate the cellular roles of these proteins, SAM, KEP1, and Who/How were expressed in mammalian and Drosophila S2 cells. KEP1 and Who/How are nuclear and SAM is cytoplasmic. The expression of KEP1 and SAM, but not Who/How, activates apoptotic pathways in Drosophila S2 cells. The identification of KEP1 and SAM implies that a large GSG domain protein family exists and helps redefine the boundaries of the GSG domain. Taken together, these data suggest that KEP1 and SAM may play a role in the activation or regulation of apoptosis and further implicate the GSG domain in RNA binding and oligomerization (Di Fruscio, 1998).

Sam68 (Src associated in mitosis, 68 kDa) is an SH3 (Src-homology 3), SH2 (Src-homology 2), and RNA binding protein that associates with and is tyrosine phosphorylated by wild-type and activated forms of c-Src in a mitosis-specific manner. Sam68 immunoprecipitated from either HeLa S3 or NIH3T3 cells is phosphorylated on threonine residues during mitosis exclusively, as well as on serine residues, during both interphase and mitosis. Recombinant Sam68, expressed as a glutathione S-transferase (GST) fusion protein, is phosphorylated on threonine and serine residues several-fold more extensively after incubation with mitotic lysates than after incubation with unsynchronized lysates. Cdc2 is identified as the kinase responsible for the mitotic threonine phosphorylation by (1) immunodepletion of the mitotic Sam68 kinase from cell lysates with anti-Cdc2 antibodies, (2) inhibition of Sam68 phosphorylation in vitro and in vivo by the cyclin-dependent kinase inhibitor olomoucine and (3) phosphorylation of Sam68 by purified Cdc2. These data demonstrate that Sam68 is a direct target of Cdc2 and may therefore mediate some of Cdc2's biological effects during mitosis (Resnick, 1997).

A natural isoform of Sam68 occurs that has a deletion within the KH domain. This isoform, called Sam68DeltaKH, is specifically expressed at growth arrest upon confluency, in normal cells. In cells that do not enter quiescence at confluency such as Src-transformed cells, no recruitment of Sam68DeltaKH is observed. Transfected Sam68DeltaKH inhibits serum-induced DNA synthesis and cyclin D1 expression. Sam68 overcomes these effects, suggesting that isoforms of Sam68 are involved, through KH domain signaling, in cell proliferation, and more precisely in G1/S transition (Barlat, 1997).

SH2/SH3 adaptor proteins are essential components of the signal transduction pathways initiated by tyrosine kinases. Nck (Drosophila homolog: Dreadlocks) is a ubiquitously expressed adaptor protein whose function has been enigmatic. Confocal microscopy was performed to localize Nck in NIH3T3 and A431 cells. Surprisingly, Nck is identified in the nucleus as well as the cytoplasm with no visible change in localization due to PDGF or EGF stimulation. There was no translocation in response to growth factor, and tyrosine phosphorylation is specific to only cytosolic Nck. Far Western blot analysis with either Nck, the SH2 domain, or the SH3 domains reveals differential binding in nuclear and cytosolic lysates, indicating specific binding partners for each subcellular location. The major target of c-Src during mitosis is SAM68, a RNA-binding protein ordinarily localized to the nucleus. SAM68 was identified as a nuclear specific binding partner of Nck in both nonmitotic and mitotic cells. Several tyrosine kinases can be found in the nucleus but their signal transduction remains undefined. The discovery of an adaptor protein in the nucleus suggests there are signal transduction mechanisms within the nucleus that recapitulate those found in the cytoplasm (Lawe, 1997).

Sam68 associates with and is tyrosine phosphorylated by Src in a mitosis-specific manner, thereby raising the possibility of a role for Src in the regulation of the cell cycle. This study examines the effects of radicicol, a Src tyrosine kinase inhibitor, on both the phosphorylation of Sam68 and mitotic progression in Src-transformed mouse fibroblasts. Radicicol reversibly inhibits the mitosis-specific tyrosine phosphorylation of Sam68 in vivo, as determined by antiphosphotyrosine immunoblotting. Radicicol inhibits the tyrosine phosphorylation of both free and Src-associated Sam68, suggesting the presence of two intracellular pools of tyrosine phosphorylated Sam68 in mitotic cells. Radicicol treatment has no effect on the ability of cells to enter mitosis, indicating that tyrosine phosphorylation of Sam68 is probably not important for cells to enter mitosis. However, radicicol reversibly retards the exit of cells from mitosis, as determined by flow cytometric analyses. Radicicol-mediated inhibition of Sam68 tyrosine phosphorylation, and its concurrent ability to block mitotic exit suggests the possibility of a significant role for Src kinase and this unique mitotic substrate, Sam68, in cell cycle regulation (Pillay, 1996).

Loops 1 and 4 of the KH domain family members are longer than the corresponding loops in other KH domains and contain conserved residues. KH domains are protein motifs that are involved in RNA binding and are often present in multiple copies. Sam68 self-associates and cellular RNA is required for the association. Deletion studies demonstrate that the Sam68 KH domain loops 1 and 4 are required for self-association. The Sam68 interaction is also observed in Saccharomyces cerevisiae by the two-hybrid system. In situ chemical cross-linking studies in mammalian cells demonstrate that Sam68 oligomerizes in vivo. These Sam68 complexes bind homopolymeric RNA and SH3 domains of p59fyn and phospholipase Cgamma1 in vitro, demonstrating that Sam68 associates with RNA and signaling molecules as a multimer. The formation of the Sam68 complex is inhibited by p59fyn, suggesting that tyrosine phosphorylation regulates Sam68 oligomerization. Other Sam68 family members including Artemia salina GRP33, Caenorhabditis elegans GLD-1, and mouse Qk1, also oligomerized. Sam68, GRP33, GLD-1, and Qk1 associate with other KH domain proteins such as Drosophila Bicaudal C. These observations indicate that the single KH domain found in the Sam68 family, in addition to mediating protein-RNA interactions, mediates protein-protein interactions (Chen, 1997).

To elucidate the biological function of Sam68, an attempt was made to identify RNA species that bind to Sam68 with high affinity, using in vitro selection. From a degenerate 40-mer pool, 15 RNA sequences were selected that bind to Sam68 with Kd values of 12-140 nM. The highest affinity RNA sequences (Kd approximately 12-40 nM) contain a UAAA motif; mutation to UACA abolishes binding to Sam68. Binding of the highest affinity ligand, G8-5, was assessed to explore the role of different regions of Sam68 in RNA binding. The KH domain alone does not bind G8-5, but a fragment containing the KH domain and a region of homology within the Sam68 subgroup of KH-containing proteins was sufficient for G8-5 binding. G8-5 binding is abolished by deletion of the KH domain or mutation of KH domain residues, analogous to loss-of-function mutations in the human Fragile X syndrome gene product and the Caenorhabditis elegans tumor suppressor protein Gld-1. These results establish that a KH domain-containing protein can bind RNA with specificity and high affinity and suggest that specific RNA binding is integral to the functions of some regulatory proteins in growth and development (Lin, 1997).

The Src family protein-tyrosine kinase, Fyn, is associated with the T cell receptor (TCR) and plays an important role in TCR-mediated signaling. A human T cell leukemia virus type 1-infected T cell line, Hayai, overexpresses Fyn. To identify the molecules downstream of Fyn, the tyrosine phosphorylation of cellular proteins was analyzed in the cells. In Hayai, a 68-kDa protein is constitutively tyrosine-phosphorylated. The 68-kDa protein coimmunoprecipitates with various signaling proteins such as phospholipase C gamma1, the phosphatidylinositol 3-kinase p85 subunit, Grb2, SHP-1, Cbl, and Jak3, implying that the protein might function as an adapter. Purification and microsequencing of this protein reveal that it is the RNA-binding protein, Sam68. Sam68 is associated with the Src homology 2 and 3 domains of Fyn and also those of another Src family kinase, Lck. CD3 cross-linking induces tyrosine phosphorylation of Sam68 in uninfected T cells. These data suggest that Sam68 participates in the signal transduction pathway downstream of TCR-coupled Src family kinases Fyn and Lck in lymphocytes (Fusaki, 1997).

Recent evidence suggests that in T lymphocytes Sam68 may act as an adaptor protein and participate in the early biochemical cascade triggered after CD3 stimulation. A direct interaction has been shown to occur between Sam68 and the two src kinases involved in T cell activation, p59(fyn) and p56(lck), as well as the partnership of Sam68 with various key downstream signaling molecules, like phospholipase Cgamma-1 and Grb2. In this study the contribution of p56(lck), as well as the role of ZAP-70, the second class of protein tyrosine kinase involved in T cell activation, were analyzed in Sam68 tyrosine phosphorylation in the human Jurkat T cell line. Using Src inhibitor PP1 and cell variants with defective expression of p56(lck), or expressing a dominant negative form of ZAP-70, it has been demonstrated that while both p56(lck) and ZAP-70 are dispensable for the low constitutive phosphorylation of Sam68 observed in Jurkat cells, a cooperation between the two kinases is required to increase Sam68's rapid phosphorylation observed in vivo after CD3 stimulation. Recombinant forms of both p56(lck) and ZAP-70 phosphorylate Sam68 in vitro. However, using CD2 stimulated cells, it has been observed that p56(lck) activation by itself does not induce Sam68 tyrosine phosphorylation. It is concluded that p59(fyn) and p56(lck) differently participate in regulating the phosphorylation state of Sam68 in T cells and that ZAP-70 may contribute to Sam68 tyrosine phosphorylation and to the specific recruitment of this molecule after CD3 stimulation (Lang, 1997).

Sam68, the 68-kDa Src substrate associated during mitosis, is an RNA-binding protein with signaling properties that contains a GSG (GRP33, Sam68, GLD-1) domain. Two Sam68-like-mammalian proteins, SLM-1 and SLM-2, have been cloned. These proteins have an ~70% sequence identity with Sam68 in their GSG domain. SLM-1 and SLM-2 have the characteristic Sam68 SH2 and SH3 domain binding sites. SLM-1 is an RNA-binding protein that is tyrosine phosphorylated by Src during mitosis. SLM-1 binds the SH2 and SH3 domains of p59fyn, Grb-2, phospholipase Cgamma-1 (PLCgamma-1), and/or p120rasGAP, suggesting it may function as a multifunctional adapter protein for Src during mitosis. SLM-2 is an RNA-binding protein that is not tyrosine phosphorylated by Src or p59fyn. Moreover, SLM-2 does not associate with the SH3 domains of p59fyn, Grb-2, PLCgamma >-1, or p120rasGAP, suggesting that SLM-2 may not function as an adapter protein for these proteins. The identification of SLM-1 and SLM-2 demonstrates the presence of a Sam68/SLM family whose members have the potential to link signaling pathways with RNA metabolism (Fruscio, 1999).

Sam68 is an RNA-binding protein that contains a heterogeneous nuclear ribonucleoprotein K homology domain embedded in a larger RNA binding domain called the GSG (GRP33, Sam68, GLD-1) domain. Members of this family of proteins are often referred to as the STAR (signal transduction and activators of RNA metabolism) proteins. It is not known whether Sam68 is a general nonspecific RNA-binding protein or whether it recognizes specific response elements in mRNAs with high affinity. Sam68 has been shown to bind homopolymeric RNA and a synthetic RNA sequence called G8-5 that has a core UAAA motif. A structure function analysis of Sam68 was performed and two arginine glycine (RG)-rich regions were identified that confer nonspecific RNA binding to the Sam68 GSG domain. In addition, by using chimeric proteins between Sam68 and QKI-7, it has been demonstrated that one of the Sam68 RG-rich sequences of 26 amino acids is sufficient to confer homopolymeric RNA binding to the GSG domain of QKI-7, another STAR protein. Furthermore, minimal sequence can also give QKI-7 the ability (as Sam68) to functionally substitute for HIV-1 REV to facilitate the nuclear export of RNAs. These studies suggest that neighboring RG-rich sequences may impose nonspecific RNA binding to GSG domains. Because the Sam68 RNA binding activity is negatively regulated by tyrosine phosphorylation, the data led to a proposal that Sam68 might be a specific RNA-binding protein when tyrosine phosphorylated (Chen, 2001).

Overexpression of Sam68 functionally substitutes for, as well as synergizes with, human immunodeficiency virus type 1 (HIV-1) Rev in RRE (Rev response element)-mediated gene expression and virus replication. In addition, COOH-terminal deletion and/or point mutants of Sam68 exhibit a transdominant negative phenotype for HIV replication. Sam68 is a member of KH domain family that includes SLM-1, SLM-2 (Sam68 like mammalian); and QKI-5, QKI-6, and QKI-7 (mouse quaking) proteins. The objective of this study was to examine the effects of these KH family proteins on RRE- and CTE (constitutive transport element of type-D retrovirus)-mediated transactivation. SLM-1 and SLM-2 proteins, which are the closest relatives of Sam68, marginally enhance RRE-mediated transactivation, while QK isoforms that are distant relatives of Sam68 have no effect. Interestingly, these proteins still enhanced the effect of Rev in RRE-mediated gene expression. The increase in chloramphenicol acetyltransferase activity is also reflected at the levels of cytoplasmic RRE-chloramphenicol acetyltransferase mRNAs, indicating that Sam68 and KH proteins may have been involved in the stability or export of unspliced RNA. The increase in Rev activity is sensitive to leptomycin B, but not to olomoucine, indicating that the effect of SLM-1, SLM-2, QKI-5, QKI-6, and QKI-7 is exerted through a CRM-1-dependent mRNA export pathway. Thus, KH family proteins play an important role in the post-transcriptional regulation of HIV (Reddy, 2002).

Other mammalian KH domain proteins

The structure of a Nova protein K homology (KH) domain recognizing single-stranded RNA has been determined at 2.4 Å resolution. Mammalian Nova antigens (1 and 2) constitute an important family of regulators of RNA metabolism in neurons, first identified using sera from cancer patients with the autoimmune disorder paraneoplastic opsoclonus-myoclonus ataxia (POMA). The structure of the third KH domain (KH3) of Nova-2 bound to a stem loop RNA resembles a molecular vise, with 5'-Ura-Cyt-Ade-Cyt-3' pinioned between an invariant Gly-X-X-Gly motif and the variable loop. Tetranucleotide recognition is supported by an aliphatic alpha helix/beta sheet RNA-binding platform, which mimics 5'-Ura-Gua-3' by making Watson-Crick-like hydrogen bonds with 5'-Cyt-Ade-3'. Sequence conservation suggests that fragile X mental retardation results from perturbation of RNA binding by the FMR1 protein (Lewis, 2000).

Mammalian splicing factor 1 (SF1; also mammalian branch point binding protein [mBBP]; hereafter SF1/mBBP) specifically recognizes the seven-nucleotide branch point sequence (BPS) located at 3' splice sites and it participates in the assembly of early spliceosomal complexes. SF1/mBBP utilizes a 'maxi-K homology' (maxi-KH) domain for recognition of the single-stranded BPS and requires a cooperative interaction with splicing factor U2AF65 bound to an adjacent polypyrimidine tract (PPT) for high-affinity binding. To investigate how the KH domain of SF1/mBBP recognizes the BPS in conjunction with U2AF and possibly other proteins, a transcriptional reporter system was constructed utilizing human immunodeficiency virus type 1 Tat fusion proteins and the RNA-binding specificity of the complex was examined using KH domain and RNA-binding site mutants. SF1/mBBP and U2AF cooperatively assemble in a reporter system at RNA sites composed of the BPS, PPT, and AG dinucleotide found at 3' splice sites, with endogenous proteins assembled along with the Tat fusions. The activities of the Tat fusion proteins on different BPS variants correlate well with the known splicing efficiencies of the variants, supporting a model in which the SF1/mBBP-BPS interaction helps determine splicing efficiency prior to the U2 snRNP-BPS interaction. Finally, the likely RNA-binding surface of the maxi-KH domain was identified by mutagenesis and appears similar to that used by 'simple' KH domains, involving residues from two putative helices, a highly conserved loop, and parts of a sheet. Using a homology model constructed from the cocrystal structure of a Nova KH domain-RNA complex, a plausible arrangement for SF1/mBBP-U2AF complexes assembled at 3' splice sites is proposed (Peled-Zehavi, 2001).


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Search PubMed for articles about Drosophila held out wings

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