Interactive Fly, Drosophila

Alternative splicing is used by metazoans to increase protein diversity and to alter gene expression during development. However, few factors that control splice site choice in vivo have been identified. Half pint (Hfp; FlyBase designation Poly-U-binding splicing factor) regulates RNA splicing in Drosophila. Females harboring hypomorphic mutations in hfp lay short eggs and show defects in germline mitosis, nuclear morphology, and RNA localization during oogenesis. hfp encodes the Drosophila ortholog of human PUF60 and functions in both constitutive and alternative splicing in vivo. In particular, hfp mutants display striking defects in the developmentally regulated splicing of ovarian tumor (otu). Furthermore, transgenic expression of the missing otu splice form can rescue the ovarian phenotypes of hfp. hfp is also required for efficient splicing of gurken mRNA and in the splicing of eukaryotic initiation factor 4E (eIF4E), which binds to the seven-methylguanosine cap at the 5' end of messenger RNAs and is a limiting factor in translation initiation (Van Buskirk, 2002).

The polytene nurse cell chromosome morphology of hfp mutants is strikingly similar to that observed in females mutant for certain alleles of ovarian tumor (otu). otu also plays a role in germline cell division, as otu egg chambers can have too few or too many germline cells. The otu transcript is subject to alternative splicing, resulting in the production of a 98 kDa protein and a less abundant 104 kDa protein (Otu104), depending on the incorporation of a 126 bp alternatively spliced exon. The biochemical functions of the Otu isoforms are unknown, but they share an N terminus that contains a cysteine protease-like domain and a C terminus that possesses weak similarity to microtubule-associated proteins. Intriguingly, the alternatively spliced exon encodes a tudor domain, a sequence element found multiply repeated in the Drosophila Tudor protein and also present in proteins with putative RNA binding functions in several species. Genetic and molecular analysis reveal distinct requirements and expression patterns for the two Otu isoforms, suggesting that the splicing of the otu transcript may be developmentally regulated. Indeed, RT-PCR shows that expression of the transcript encoding the 104 kDa isoform is restricted to the early stages of oogenesis (Van Buskirk, 2002).

In order to determine whether the alternative splicing of otu is dependent on hfp function, the expression of otu was examined in hfp mutants. RT-PCR analysis of RNA isolated from early stage egg chambers shows that the relative abundance of the larger otu transcript is decreased in hfp, and Western analysis shows that the levels of the corresponding 104 kDa Otu isoform are significantly decreased. Severe loss of Otu104 activity results in the production of tumorous egg chambers, which are not observed in hfp. Therefore, some residual Otu104 protein must be present, possibly because the hfp mutants represent partial loss-of-function alleles. To determine which, if any, of the hfp phenotypes are due to an effect on otu splicing regulation, a hybrid genomic-cDNA transgene encoding exclusively the 104 kDa Otu isoform was introduced into an hfp mutant background. hfp mutants expressing two copies of the Otu104 transgene produce egg chambers with predominantly wild-type nurse cell nuclear morphology and expression of four copies of the transgene completely rescues the nurse cell and oocyte nuclear morphology as well as the germline division defect. Expression of Otu104 also restores normal grk mRNA localization. This was unexpected, since defects in grk RNA localization have not been previously described in otu mutants, and no grk RNA localization defects are observed in otu⁷ homozygotes or otu⁷/otu¹¹ mutants, which give rise to differentiated egg chambers. However, otu¹¹ females, though often producing tumorous germaria, will under certain conditions produce rare eggs, and these show defects, including the expansion of dorsal appendages associated with mislocalization of grk RNA, very similar to those of hfp⁹ mutant eggs. Interestingly, otu¹¹ is a point mutation in the alternatively spliced exon and hence specifically affects the activity of the 104 kDa Otu isoform (Van Buskirk, 2002).

Hrb27C, Sqd and Otu cooperatively regulate gurken RNA localization and mediate nurse cell chromosome dispersion in Drosophila oogenesis

Heterogeneous nuclear ribonucleoproteins, hnRNPs, are RNA-binding proteins that play crucial roles in controlling gene expression. In Drosophila oogenesis, the hnRNP Squid (Sqd) functions in the localization and translational regulation of gurken (grk) mRNA. Sqd interacts with Hrb27C, an hnRNP previously implicated in splicing. Like sqd, hrb27C mutants lay eggs with dorsoventral defects and Hrb27C can directly bind to grk RNA. These data demonstrate a novel role for Hrb27C in promoting grk localization. A direct physical interaction is also observed between Hrb27C and Ovarian tumor (Otu), a cytoplasmic protein implicated in RNA localization. Some otu alleles produce dorsalized eggs and it appears that Otu cooperates with Hrb27C and Sqd in the oocyte to mediate proper grk localization. All three mutants share another phenotype, persistent polytene nurse cell chromosomes. These analyses support dual cooperative roles for Sqd, Hrb27C and Otu during Drosophila oogenesis (Goodrich, 2004).

This study identifies a physical and genetic interaction between Hrb27C and Sqd. The two proteins were previously biochemically purified as part of an hnRNP complex from Drosophila cells, but it was not known if they interact directly nor had their RNA targets been isolated. The interaction was originally detected in a two-hybrid screen and it has been confirmed biochemically by co-immunoprecipitation. In vivo, this interaction requires the presence of RNA; this result was unexpected because the yeast two-hybrid constructs that originally revealed the interaction did not contain the RRMs and presumably cannot bind RNA. The interaction may require the full-length proteins to be in unique conformations that are achieved only when they are bound to RNA. The truncated proteins used in the yeast two-hybrid screen may be folded in such a manner that the protein-protein interaction domains are exposed even in the absence of RNA binding, making it possible for the interaction to occur in yeast (Goodrich, 2004).

A striking genetic interaction was detected between Sqd and Hrb27C; weak hrb27C mutants can strongly enhance the DV defects of weak sqd mutants. This suggests that the physical interaction has in vivo significance. The phenotypes of sqd and hrb27C place both proteins in a pathway required for proper grk localization and suggest a previously undetected role for Hrb27C. hrb27C mutants lay variably dorsalized eggs, and in situ hybridization analysis reveals that grk RNA is mislocalized. Since the mislocalized RNA in hrb27C mutants is translated, causing a dorsalized egg phenotype, it is likely that Hrb27C also functions with Sqd in regulating Grk protein accumulation. A role in the regulation of RNA localization and translation is novel for Hrb27C/Hrp48; it has been previously described as functioning in the inhibition of IVS3 splicing of P-element encoded transposase (Siebel, 1994; Hammond, 1997). Hrb27C has nuclear functions and has been observed in the nucleus and cytoplasm of somatic and germline cells in embryos (Siebel, 1995). Because Sqd is localized to the oocyte nucleus where it is thought to bind grk RNA, a model is favored where Hrb27C and Sqd bind grk RNA together and are exported possibly as a complex into the cytoplasm (Goodrich, 2004).

Two unpublished studies have implicated Hrb27C in osk RNA localization (J. Huynh, T. Munro, K. Litière-Smith and D. St Johnston, communication to Goodrich, 2004) and translational regulation (T. Yano, S. Lopez de Quinto, A. Shevenchenko, A. Shevenchenko, T. Matsui and A. Ephrussi, communication to Goodrich, 2004). Translational repression of osk RNA until it is properly localized is essential to prevent disruptions in the posterior patterning of the embryo. An analogous mechanism of co-regulation of mRNA localization and translation appears to control Grk expression. Certain parallels between osk and grk regulation are very striking. Both RNAs are tightly localized and subject to complex translational regulation; they also share certain factors that mediate this regulation. In addition to Hrb27C, the translational repressor Bruno appears to be a part of the regulatory complexes that are required for the proper expression of both RNAs. It will be interesting to determine if there are other shared partners that function in the regulation of both RNAs, as well as identify the factors that give each complex its localization specificity (Goodrich, 2004).

Otu is also involved in the localization of grk mRNA and interacts physically with Hrb27C. Though a definitive role of Otu in regulating Grk expression has not been previously described, Van Buskirk (2002) found that four copies of Otu104 (flies carry a transgene expressing only the 104 kDa isoform of Otu under the control of the otu promoter) are able to rescue the grk RNA mislocalization defect of half pint (hfp; poly U binding factor 68kD) mutants. Analysis of the hypomorphic alleles, otu⁷ and otu¹¹, reveals a requirement for Otu in localizing grk RNA for proper DV patterning. Alternative splicing of the otu transcript produces two protein isoforms: a 98 kDa isoform and a 104 kDa isoform that differ by the inclusion of a 126 bp alternatively spliced exon (6a) in the 104 kDa isoform. This alternatively spliced exon encodes a tudor domain, a sequence element present in proteins with putative RNA-binding abilities. Interestingly, the otu¹¹ allele, which contains a missense mutation in exon 6a, shows the grk localization and dorsalization defect, strongly suggesting that the tudor domain of Otu plays an important function in grk localization. Since otu mutants also show defects in osk localization, it appears that like several other factors, Otu is required for both grk and osk localization. Additionally, Otu has been isolated from cytoplasmic mRNP complexes (Goodrich, 2004).

Surprisingly, an additional shared phenotype of hrb27C, sqd and otu mutants was found. During early oogenesis, the endoreplicated nurse cell chromosomes are polytene. As they begin to disperse, the chromosomes are visible as distinct masses of blob-like chromatin that completely disperse by stage 6. The formation of polytene nurse cell chromosomes is due to the endocycling that occurs during early oogenesis. The mechanism of chromosome dispersal is hypothesized to involve the degradation of securin by the anaphase-promoting complex/cyclosome as a result of separase activity that cleaves cohesin. The significance of chromosome dispersion is not clear, but it has been suggested that it could facilitate rapid ribosome synthesis. A defect in the dispersal of polytene chromosomes has been previously described for otu mutants, and both sqd and hrb27C mutants also have nurse cell chromosomes that fail to disperse. Weak double transheterozygous allele combinations of sqd and hrb27C produce the persistent polytene nurse cell chromosome phenotype that is not observed in either of the weak mutants alone. Thus, genetic and physical interactions between these gene products suggest that they function together in regulation of this process (Goodrich, 2004).

The failure to disperse nurse cell chromosomes has also been observed in mutants of hfp where the nurse cell nuclear morphology defect is due to improper splicing of Otu104 (Van Buskirk, 2002). As was shown for hfp, expressing Otu104 in a sqd mutant background is able to rescue the polytene nurse cell chromosome phenotype. Although Hfp affects otu splicing, it is not clear how Sqd functions to mediate this phenotype, but evidence supports a role in Otu104 protein accumulation. The transcript that encodes Otu104 is clearly present in sqd mutants, as assayed by RT-PCR, but the level of Otu104 protein is decreased. These results suggest that the level of Otu104 is crucial to maintaining proper nurse cell chromosome morphology because the polytene phenotype of sqd mutants can be rescued by expressing only one extra copy of Otu104 and the majority of egg chambers from females heterozygous for otu¹³, otu¹¹, or a deficiency lacking otu, have nurse cell chromosomes that fail to disperse (Goodrich, 2004).

The reduced level of Otu104 in sqd mutants raises the possibility that Sqd could translationally regulate otu RNA and that the polytene nurse cell phenotype may be a result of the decreased level of Otu104. Alternatively, Sqd and Hrb27C could form a complex that recruits and stabilizes Otu104 protein, and this complex in turn functions directly or indirectly to regulate chromosome dispersion. An indirect role seems more likely, given that Otu has never been observed in the nucleus. Since Otu104 can rescue the phenotype of sqd mutants and Otu and Hrb27C co-precipitate, it seems plausible that Sqd and Hrb27C interact with Otu in the nurse cell cytoplasm to affect an RNA target that could then mediate chromosome dispersion (Goodrich, 2004).

The genetic data support a model in which Sqd, Hrb27C, and Otu function together in a complex that affects at least two processes during oogenesis: DV patterning within the oocyte and mediation of nurse cell chromosome dispersion. Although the biochemical data are consistent with this model, it is also possible that Hrb27C and Sqd could form a complex that is distinct from a complex containing Hrb27C and Otu. However, the in vivo genetic interactions and mutant phenotypes reveal that all three proteins affect both processes and favor a model in which all three proteins function in one complex (Goodrich, 2004).

These results allow expansion upon the model proposed by Norvell (1999) for the regulation of grk RNA expression. Sqd and Hrb27C associate with grk RNA in the oocyte nucleus. Hrb27C and Sqd remain associated with grk in the cytoplasm where Otu and possibly other unidentified proteins associate with the complex as necessary to properly localize, anchor and translationally regulate grk RNA. A likely candidate to be recruited to the complex is Bruno, which interacts with Sqd (Norvell, 1999). Although the RNA target of Sqd, Hrb27C and Otu in the nurse cells is not known, the proteins may form a complex composed of different accessory factors to regulate localization and/or translation of RNAs encoding proteins that affect chromosome morphology (Goodrich, 2004).

Although Hrb27C, Sqd and Otu function together to affect different processes in the oocyte and the nurse cells, they probably function to mediate the precise spatial and temporal regulation of a target RNA that is unique to each cell type where the presence of additional factors would provide specificity to each complex. The importance of the precise localization and translational regulation of grk is well defined, and Hrb27C and Otu have not been identified as additional proteins that facilitate these processes. The transition from polytene to dispersed chromosomes in the nurse cells is less well characterized, but most probably requires precise spatial and temporal regulation as well. A complex containing Sqd, Hrb27C and Otu could function to ensure that the target RNA is properly localized and functional in the nurse cells only at the proper stage of development to promote chromosome dispersal (Goodrich, 2004).

Otu in germ-line sex determination

Continued: see Regulation part 2/2

ovarian tumor: Biological Overview | Developmental Biology | Effects of Mutation | References

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