InteractiveFly: GeneBrief

crooked neck: Biological Overview | References

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 (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).

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, 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).

Reference names in red indicate recommended papers.

Search PubMed for articles about Drosophila Crooked neck

Burnette, J. M., Hatton, A. R. and Lopez, A. J. (1999). Trans-acting factors required for inclusion of regulated exons in the Ultrabithorax mRNAs of Drosophila melanogaster. Genetics 151: 1517-1529. PubMed ID: 10101174

Edenfeld, G., et al. (2006). The splicing factor crooked neck associates with the RNA-binding protein HOW to control glial cell maturation in Drosophila. Neuron 52(6): 969-80. PubMed ID: 17178401

Gasch, A., et al. (2005). The structure of PRP40 FF1 domain and its interaction with the CRN-TPR1 motif of CLF1 gives a new insight into the binding mode of FF domains. J. Biol. Chem. 281: 356-364. PubMed ID: 16253993

Park, J. W., et al. (2004). Identification of alternative splicing regulators by RNA interference in Drosophila, Proc. Natl. Acad. Sci. 101: 15974-15979. PubMed ID: 15492211

Raisin-Tani, S. and Leopold, P. (2002). Drosophila crooked-neck protein co-fractionates in a multiprotein complex with splicing factors. Biochem. Biophys. Res. Commun. 296: 288-292. PubMed ID: 12163015

Volohonsky, G., Edenfeld, G., Klambt, C. and Volk, T. (2007). Muscle-dependent maturation of tendon cells is induced by post-transcriptional regulation of stripeA. Development 134(2): 347-56. PubMed ID: 17166919

Wang, Q., Hobbs, K., Lynn, B. and Rymond, B. C. (2003). The Clf1p splicing factor promotes spliceosome assembly through N-terminal tetratricopeptide repeat contacts. J. Biol. Chem. 278: 7875-7883. PubMed ID: 12509417

Zhang, K., Smouse, D. and Perrimon, N. (1991). The crooked neck gene of Drosophila contains a motif found in a family of yeast cell cycle genes. Genes Dev. 5: 1080-1091. PubMed ID: 2044955

date revised: 25 September 2007

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