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

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).


Two upstream exons are separated by a large intron, at least 20 kb long, from the remainder of the exons that contain the majority of the open reading frame sequences. Sequence identity between the two transcripts extends until just before the end of the open reading frame of the 4.0 kb cDNA. Additional sequencing of the 4.5 kb cDNA reveals that the C-terminal portion of the longer transcript codes for an alternative 6-amino-acid C-terminal tail that replaces the final 36 amino acids of the open reading frame of the 4.0 kb cDNA (Lo, 1997). This type of alternative splicing to produce proteins with divergent C-termini is similar to that seen in the mouse quaking gene, where alternative splicing produces three different mRNAs, resulting in proteins with three alternative carboxy tails (Ebersole, 1996).

Genomic length - 35 kb

cDNA clone length - 4.0 kb or 3.6 kb (maternal) and 4.5 (zygotic)

Bases in 5' UTR - 317

Bases in 3' UTR - 8

Bases in 3' UTR - approximately 2.5 kb


Amino Acids - 394 (maternal) and 407 (zygotic)

Structural Domains

The mouse quaking gene product and the C. elegans gld-1 gene display high degrees of similarity to the how gene in a region including the KH domain. Originally defined as an about 50-amino-acid-long motif predicted to have a beta-alpha-alpha-beta-beta secondary structure, the KH domain has recently been redefined as the maxi-KH domain to include an additional alpha-helix of about 18 amino acids at the carboxy end that was discovered in structural studies (Musco, 1996). Within the maxi-KH domain, How protein is 79% and 73% identical to Quaking and Gld-1 proteins, respectively. These three proteins are members of a small subfamily of single KH-domain proteins that have a unique, strongly conserved loop between the beta2 and beta3 strands of the domain. Other members of this subfamily include the Artemia glycine-rich protein GRP33, the mammalian splicing factor SF, and the Src-associated during mitosis protein Sam68, found in both mouse and human. An additional, smaller region of homology, about 25 amino acids, carboxy-terminal to the maxi-KH domain called the CGA region (Jones, 1995) is shared by the How, Quaking, Gld-1 and SF1 proteins. This region could also contain SH2-binding sites, as in Sam68. The other prominent feature of the How protein is an alanine- (15 residues) and glutamine- (31 residues) rich region from amino acids 1-75 that is characteristic of a number of Drosophila transcription factors.

Members of the subfamily of KH-domain-containing proteins, which appear to link a signal transduction pathway to RNA metabolism, have been named Signal Transduction and Activation of RNA (STAR) proteins (Ebersole, 1996). In this subfamily, the proteins contain only one KH-domain; members included are the Sam68 mouse protein and its human homolog, p62, which plays a role during mitosis, and C. elegans Gld-1, which behaves as a tumor suppressor gene in the germ line (Baehrecke, 1997; Lo, 1997; Zaffran, 1997 and references).

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

date revised: 30 March 2002

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