Protein interactions, transport and localization of transcripts

The Drosophila gene squid (sqd) encodes a heterogeneous nuclear RNA binding protein (hnRNP), also known as hrp40. hnRNPs constitute a large family of proteins that has been implicated in the processing of nascent mRNA transcripts. Recent studies have demonstrated that a subset of hnRNPs, including human hnRNP A1 and A2, Saccharomyces cerevisiae Nplp3 and Hrp1, and Chironomus tentans Hrp36, rapidly shuttle between the cytoplasm and the nucleus. A specific motif, termed M9, has been shown to mediate this nucleocytoplasmic shuttling, and this motif is present in Sqd. Nuclear import of M9-containing hnRNPs is achieved by an association with the nuclear import protein Transportin. Studies of several of these hnRNPs have indicated that one of their major roles is the nuclear export of mRNAs, suggesting that Sqd may perform a similar function during Drosophila oogenesis (Norvell, 1999 and references).

Sqd protein is detected within the somatic follicle cells and the germ-line-derived nurse cells and oocyte. A germ-line mutation in a squid causes female sterility as a result of mislocalization of Gurken (GRK) mRNA during oogenesis. Alternative splicing produces three isoforms: SqdA, SqdB, and SqdS. These isoforms are not equivalent: SqdA and SqdS perform overlapping but nonidentical functions in GRK mRNA localization and protein accumulation, whereas SqdB cannot perform these functions. Furthermore, although all three Sqd isoforms are expressed in the germline cells of the ovary, they display distinct intracellular distributions. Both SqdB and SqdS are detected in germ-line nuclei, whereas SqdA is predominantly cytoplasmic. It is argued that SqdS is involved in the transport and localization of GRK mRNA. The ability of SqdA to prevent translation of ventrally localized GRK mRNA and its ability to provide peak levels of Grk protein required on the dorsal side of the egg chamber, strongly suggests that SqdA has a role in the accumulation of Grk protein. Moreover, the role of SqdA is both positive and negative, suggesting that SqdA may influence the association of GRK mRNA with appropriate translational regulators (Norvell, 1999).

Evidence is provided that GRK mRNA localization and translation are coupled by an interaction between Sqd and the translational repressor protein Bruno. Because Sqd protein binds GRK mRNA directly and belongs to the class of hnRNPs implicated in nuclear mRNA export, it seems likely that Sqd functions in the nuclear export of GRK mRNA. One complication in this model is that in the sqd mutant, GRK mRNA is still able to leave the nucleus and accumulate in the oocyte cytoplasm but not in the dorso-anterior corner. Therefore, the function of the Sqd protein appears to be in the regulated nuclear export of GRK mRNA, such that Sqd is responsible for delivering the GRK message to a cytoplasmic protein involved in its anchoring, possibly coupled to GRK translation. Thus, one might expect that Sqd protein should interact with some cytoplasmic ovarian proteins (Norvell, 1999).

A number of proteins have been implicated in the translational regulation of GRK mRNA, both positively (e.g., Encore and Vasa) and negatively (e.g., Bruno). Therefore, an investigation was carried out to see whether Sqd protein could directly associate with any of these candidates. Using the in vitro association assay, interactions were sought between the Sqd isoforms and Encore, Vasa, or Bruno. Although a direct interaction between Sqd and Encore or Vasa could not be found, Sqd protein associates with Bruno protein in vitro. Although the Sqd-Bruno interaction observed in vitro is not extremely strong, it is very consistently observed over multiple experiments. These data show that Sqd protein and Bruno protein can associate with one another. Moreover, these data provide evidence for a link between GRK mRNA localization and translational regulation (Norvell, 1999).

At least two lines of evidence indicate that Sqd protein must be present within the oocyte nucleus for GRK mRNA to be localized properly during oogenesis. (1) Of the three Sqd isoforms, only SqdS is detected within the oocyte nucleus; among the Sqd transgenic females, only those expressing the SqdS isoform show properly localized GRK mRNA. The differential nuclear accumulation of the SqdS protein is associated with its ability to interact with Drosophila Transportin. SqdA does not possess a Transportin interaction motif. (2) The relationship between K10 and the distribution of Sqd protein also demonstrates the importance of Sqd accumulation within the oocyte nucleus (Norvell, 1999).

Nuclear import of M9-containing hnRNPs is mediated through an interaction with the import protein Transportin. Because the M9 motif is expressed differentially in the Sqd isoforms, it seemed possible that the observed differences in nuclear accumulation could be attributable to a differential association with Transportin. To address this issue, a study was made of the ability of in vitro transcribed and translated Drosophila Transportin protein to bind to GST fusion proteins corresponding to the Sqd isoforms. In direct correlation to the intracellular distributions observed, it was found that Transportin protein associates strongly with both SqdS and SqdB (the two isoforms detected within germ-line nuclei), but much less strongly with SqdA, which assumes a cytoplasmic distribution. At this time it is not clear why SqdA and SqdB, which share the same M9-like motif, behave so differently with respect to nuclear accumulation and association with Transportin. The carboxyl termini of these two isoforms are different from one another, with SqdB having an extensively glycine-rich domain that SqdA does not contain. In addition, the reason for the exclusive accumulation of SqdS, and not SqdB, within the oocyte nucleus is not known. One potential explanation could be that SqdS may be transported preferentially into the oocyte, thus allowing its import into the oocyte nucleus, whereas SqdB may be retained in the nurse cells. In any event, these data demonstrate that the differential nuclear accumulation of the Sqd proteins is associated with a differential ability to interact with Drosophila Transportin (Norvell, 1999).

Sqd protein can interact with GRK mRNA. The sqd gene is clearly required for proper GRK mRNA localization. Since it encodes a known RNA-binding protein, one potential target for Sqd is GRK mRNA itself. Therefore, a UV cross-linking analysis was undertaken to look for interactions between ovarian proteins and parts of the GRK RNA. In these experiments, wild-type ovarian extracts were used as a source of Sqd protein. Different regions of the GRK 3' untranslated region (UTR) were used as probes, and as a negative control, the +3 fragment of the Nanos (NOS) 3' UTR was used, because Nanos mRNA localization is unaffected by the sqd1 mutation. To investigate whether Sqd protein can interact with the 3' UTR of the GRK transcript, ovarian extracts were incubated with labeled GRK 3' UTR fragment 1 or the nos +3 fragment. The appropriately treated protein-RNA complexes were subjected to immunoprecipitation with a monoclonal anti-Sqd antibody and visualized after SDS-PAGE. The Sqd proteins, which are resolved as a distinct doublet at ~42 kD, interact well with the 3' UTR of GRK. On the basis of molecular mass, and the immunoprecipitation experiments, it appears that the 42-kD bands are Sqd protein (Norvell, 1999).

The ability to investigate the roles of the individual Sqd isoforms in the regulation of Grk during Drosophila oogenesis has revealed that there are two key aspects of Grk regulation: GRK mRNA localization and Grk protein accumulation. Both of these critical aspects of Grk regulation are accomplished by Sqd protein; however, these functions are performed differentially by the SqdS and SqdA isoforms. The severity effects of sqd mutation, therefore, reflect the loss of function of both of these proteins within the germ line, thus causing the mislocalization of GRK mRNA and the inappropriate accumulation of ectopic Grk protein. Restoration of either of these levels of regulation allows partial rescue of the D-V patterning defects of sqd mutants, but full rescue requires the function of both SqdS and SqdA. The data suggest that Sqd protein is a key regulator of both aspects of Grk regulation. The interaction of Sqd with the translational repressor protein Bruno provides a link between GRK mRNA localization and its translational regulation. Bruno has been shown to have a direct role in the translational repression of unlocalized Oskar mRNA. The interaction between Bruno and OSK mRNA is mediated by a specific sequence within the OSK message, which is termed BRE. As of yet, the molecular mechanism of Bruno action is not fully understood. However, correctly localized OSK mRNA must somehow be relieved from the Bruno-mediated repression by specific trans-acting factors localized to the posterior of the embryo (Norvell, 1999).

The interaction between Sqd and Bruno suggests that Bruno may play a role in the translational regulation of GRK mRNA. In support of this, GRK mRNA is known to contain a BRE within its 3' UTR, and Bruno has been shown to bind the GRK message. In addition, it has been demonstrated that during late stages of oogenesis, Bruno protein is concentrated at the anterior end of the oocyte, in a position that is coincident with localized GRK mRNA. On the basis of protein interaction data, it is suggested that Bruno may serve as a translational repressor of unlocalized GRK mRNA. As is the case for localized OSK mRNA, the appropriately localized GRK message would be relieved of its Bruno-mediated repression by other localized trans-acting factors. Moreover, the physical association between Sqd protein and Bruno protein suggests an appealing model to explain the similarity of the sqd and K10 mutant phenotypes. These two female sterile mutations represent the only cases in which mislocalized GRK mRNA is translated consistently and efficiently in all egg chambers. It is proposed that the role of Sqd protein is to take GRK mRNA from the oocyte nucleus, recruit Bruno in the cytoplasm, and deliver GRK mRNA to an anchor. In the absence of nuclear Sqd, in either sqd or K10 egg chambers, GRK RNA exits the nucleus by a generalized export mechanism, but does not associate efficiently with the repressor Bruno nor with the anchor. Because the interaction between GRK mRNA and Bruno does not occur, even the unlocalized GRK mRNA is translated efficiently. Using this model, Sqd protein provides the physical link between GRK mRNA transport, localization, and its appropriately regulated translation (Norvell, 1999).

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 on 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).

Fs(1)K10, a protein than interacts with Squid

Mutations in both fs(1)K10 and sqd, consistently cause a mislocalization of GRK mRNA along the entire anterior cortex of the oocyte and lead to the production of strongly dorsalized eggs. Although the nuclear import of SqdS protein is most likely driven by its association with Transportin, K10 function is required for the stable accumulation of Sqd in the oocyte nucleus. This places K10 function upstream of Sqd in the germ line. Accumulation of Sqd protein in the nurse cells is not affected, and in addition, Sqd is detectable within the oocyte cytoplasm of K10 mutants. Since the SqdS protein is the only Sqd isoform that is normally detected within the oocyte nucleus, the major effect of K10 must be on the nuclear retention of SqdS (Norvell, 1999 and references).

At this time it is unclear how K10 performs this function mechanistically. K10 protein will physically interact with the Sqd isoforms. The only known motif within the K10 protein is a potential helix-turn-helix domain in the carboxyl terminus, but site-directed mutagenesis of this domain has revealed that this motif is unnecessary for K10 function. K10 could be responsible for the modification of Sqd protein in such a manner as to promote nuclear retention, or alternatively, K10 could form a complex with Sqd protein that stabilizes its accumulation within the oocyte nucleus. In either case, the finding that Sqd protein requires the presence of K10 to accumulate in the oocyte nucleus further suggests that the phenotype of K10 mutant eggs is attributable to an effect on Sqd (Norvell, 1999 and references).

The establishment of the major body axes of the Drosophila egg and future embryo requires strict regulation of Gurken mRNA and protein localization. GRK mRNA and protein localization is dependent on synthesis of GRK transcripts in the oocyte nucleus and on RNA localization elements in the 5' portion of the transcript. Gurken mRNA and protein localization is dependent on region-specific translation of Gurken transcripts and K10 is identified as a probable negative regulator of Gurken translation (Saunders, 1999).

A critical step in Drosophila dorsoventral patterning is the movement of Gurken mRNA from the anterior cortex of the oocyte to the oocyte's anterodorsal corner at stage 8 of oogenesis. Such movement is dependent on fs(1)K10. It has been proposed that fs(1)K10 mediates Gurken mRNA movement by down-regulating Gurken mRNA levels, thus ensuring that Gurken mRNA does not saturate its receptors located in the oocyte's anterodorsal corner. In contradiction to this model, it has been shown, both genetically and immunocytochemically, that Grk protein levels are lower in the anterodorsal region of fs(1)K10 mutant oocytes than in the anterodorsal region of fs(1)K10+ oocytes. From this and other data, a more direct role for fs(1)K10 in the Gurken mRNA localization process is proposed (Serano, 1995).

Splicing of Squid mRNA

Direct interactions between RNA-binding proteins and snRNP particles modulate eukaryotic pre-mRNA processing patterns to control gene expression. The conserved U1 snRNP-interacting RNA-binding protein PSI is essential for Drosophila viability. A null PSI mutation is recessive lethal at the first-instar larval stage, and lethality is fully rescued by transgenes expressing the PSI protein. A mutant transgene that lacks the PSI-U1 snRNP-interaction domain restores viability but shows courtship behavior abnormalities and meiosis defects during spermatogenesis, resulting in a complete male sterility phenotype. Using cDNA microarrays, specific target mRNAs have been identified with altered expression profiles in these mutant males. A subset of these transcripts is also found associated with PSI in endogenous immunopurified ribonucleoprotein complexes. One specific target, the hrp40/squid transcript, shows an altered pre-mRNA splicing pattern in PSI mutant testes. It is concluded that a functional association between the PSI protein and the spliceosomal U1 snRNP particle is required for normal Drosophila development and for the processing of specific PSI-interacting cellular transcripts. These results also validate the use of cDNA microarrays to characterize in vivo RNA-processing defects and alternative pre-mRNA splicing patterns (Labourier, 2002).

It was asked whether PSI differentially associates with specific mRNAs in endogenous ribonucleoprotein complexes. PSI-interacting RNAs were identified by large-scale immunoaffinity purification using anti-PSI polyclonal antibodies and hybridization to Drosophila cDNA microarrays. Analysis of five independent microarrays allowed for a characterization of the transcripts that were reproducibly present in PSI-containing ribonucleoprotein particles isolated from Canton-S embryonic nuclear extracts. Interestingly, ~20% of these transcripts, such as yps, Hsp60, and desat1, were also differentially expressed in males mutant for PSI (v16;P[PSIDeltaAB]). RT-PCR experiments confirmed that these mRNAs are highly enriched in immunopurified PSI fractions but not in control immunopurifications using total rabbit IgG and embryonic nuclear extracts. In addition, each of the tested mRNAs shows the expected three- to two-fold increased (Taf60-2 and Hsp60) or decreased (yps, desat1, and plexA) level of expression in v16;P[PSIDeltaAB] sterile males versus v16;P[PSI] wild-type males (Labourier, 2002).

Since no variation was observed for other control mRNAs, it is concluded that the loss of PSI-U1snRNP interaction in v16;P[PSIDeltaAB] males can result in an incorrect processing of specific PSI-interacting transcripts (Labourier, 2002).

Among the specific targets that are reproducibly highly ranked in both the mRNA profiling and PSI-interacting databases is squid (CG17791). squid encodes a heterogeneous nuclear RNA-binding protein (hnRNP); a squid germ-line mutation causes female sterility. Furthermore, the squid pre-mRNA is alternatively spliced to produce three protein isoforms designated SqdA (hrp40.1), SqdS (hrp40.2), and SqdB that each perform different functions in the female germ line. Strikingly, the EST detected in the microarrays experiments (LD09564) was specific for the unspliced SqdA isoform (Labourier, 2002).

RT-PCR analyses show that the SqdA mRNA is the major form in embryonic extracts and is present in PSI-containing ribonucleoprotein complexes. In adult males, all of the three transcripts are expressed, although the SqdA mRNA is five times more abundant than SqdB, and the SqdS isoform is barely detectable. A significant reduction of expression in v16;P[PSIDeltaAB] males is observed only for the unspliced SqdA mRNA. By quantification of these PCR products, it was determined that the spliced/unspliced (SqdB/SqdA) ratio increases by twofold in v16;P[PSIDeltaAB] males, suggesting that the PSIDeltaAB protein is less effective at inhibiting SqdB splicing. Similarly, a reduced level of SqdA mRNA expression is detected in testes dissected from v16;P[PSIDeltaAB] males. In contrast, the squid splicing profile is very different in the female germ line. RT-PCR analyses indicate that the accurately spliced SqdB isoform is highly expressed in ovaries dissected from v16;P[PSI] females. Consistent with the observation that v16;P[PSIDeltaAB] females do not exhibit oogenesis defects, no variation of the SqdB/SqdA ratio was detected between v16;P[PSI] and v16;P[PSIDeltaAB] ovaries. Taken together, these data suggest that a direct interaction between PSI and U1 snRNP particles is required in vivo for an efficient tissue-specific splicing inhibition of a subset of PSI-interacting transcripts such as the hrp40/squid pre-mRNA (Labourier, 2002).

PSI was initially identified as a soma-specific pre-mRNA splicing factor, detected only at very low level in Drosophila ovaries by immunoblot analyses. The above results prompted a determination of whether PSI is expressed in Drosophila testes. High levels of PSI and PSIDeltaAB proteins were detected by immunoblot analysis in testes dissected from w1118, v16;P[PSI], or v16;P[PSIDeltaAB] mature males. Immunostaining of whole-mount testes shows that both somatic and germ-line cells are stained by anti-PSI polyclonal antibodies at the apical end of w1118 or v16;P[PSI] adult testes. Only PSI-positive somatic cells were observed in the basal and central part of the testes since spermatids, and mature sperm did not stain. Expression of PSI in germ cells was further observed in w1118 or v16;P[PSI] males by double-labeling with anti-PSI and anti-vasa antibodies, and in v16;P[PSIDeltaAB#1] males, where excess, premeiotic, vasa-positive spermatocytes were also strongly stained by anti-PSI antibodies throughout the testes. Importantly, both the PSI and PSIDeltaAB proteins were localized to the nucleus and expressed at high levels in somatic and germ-line cells (Labourier, 2002).

In contrast, the pattern of PSI expression in ovaries is very different. Only very low levels of PSI expression were detected by confocal imaging in the nuclei of the somatically derived follicle cells. A weak staining was also observed in nurse cell nuclei during the early stages of oogenesis. Consistent with this localization, in situ hybridization using a digoxygenin-labeled anti-sense RNA probe shows that the PSI mRNA is expressed in the nurse cell and early oocyte cytoplasm, as well as in the follicle cells surrounding the mature oocyte. This pattern suggests that translation of PSI mRNA is down-regulated in the female germ line, because the level of PSI protein is low in nurse cells and oocytes. Furthermore, an FLP-DFS-induced germ-line clone analysis showed that PSI is not essential for oogenesis. v16/v16 mosaic females laid embryos without obvious defects that developed into normal adult flies. It is concluded that unlike in the female gonad, PSI is highly expressed in the male germ line, where its function is required during spermatogenesis (Labourier, 2002).

This work provides several new insights into how RNA-binding proteins contribute to the control of gene expression patterns and the execution of underlying developmental programs in metazoans. (1) It has been show that the KH-type RNA-binding protein PSI has a crucial cellular function required for Drosophila viability. PSI is a nuclear protein widely expressed throughout fly development, and a PSI null mutation is lethal at the first-instar larval stage. (2) Specific target transcripts that interact with and are regulated by PSI in vivo were identified. (3) It was shown that the PSI C-terminal AB motif, which mediates the interaction of PSI with endogenous U1 snRNP particles, is essential for normal Drosophila development. Transgenic flies lacking the PSI AB motif exhibit a male sterility phenotype. (4) Evidence has been presented that loss of the PSI-U1snRNP association affects the processing of a subset of PSI-interacting transcripts in vivo. These findings extend previous studies showing an involvement of PSI in the regulation of P-element transposase expression and clarify an understanding of the function(s) of KH-domain-containing proteins during metazoan development (Labourier, 2002).

Biochemical and genetic experiments have shown that PSI contributes to the soma-specific inhibition of P-element pre-mRNA splicing by binding to P-element exonic sequences and by interacting, through its AB motif, with the Drosophila U1 snRNP 70K protein. This protein-protein interaction is very specific and, so far, no other PSI-interacting proteins or proteins containing an AB-like motif have been identified in Drosophila. The isolation of the recessive lethal v16 PSI mutant allele allows for the examination of the contribution to metazoan development of a direct and specific interaction between a nuclear RNA-binding protein and a constitutive snRNP particle. Although full-length PSI protein is required to fully complement the v16 mutation, transgenic Drosophila in which the only source of PSI protein is the DeltaAB domain deletion mutant are viable. This suggests that the four PSI KH domains are necessary and sufficient to rescue Drosophila somatic development. The PSI AB motif does not contribute to the specificity and affinity of PSI RNA binding in vitro. Thus, the KH domains of PSI may be sufficient to target PSI to pre-mRNA containing high-affinity sequences during specific developmental stages or in specific tissues. In addition, a functional interaction between PSI and U1 snRNP particles, mediated by the PSI AB motif, may be crucial to control the processing of specific PSI-interacting transcripts, essential for a subset of tissue-, stage-, or sex-specific developmental programs (Labourier, 2002).

Consistent with this idea, it was found that transgenic v16;P[PSIDeltaAB] males are completely sterile, but their rescued sisters are fertile and lay eggs without apparent abnormalities. Although little data are available, somatic components of the male gonad seem to communicate with the germ line to influence its development. Somatic signals have been suggested to control the self-renewing potential of male germ-line stem cells during spermatogenesis in mammals and Drosophila. The observation that PSI is highly expressed in both germ-line and somatic cells in testes invites speculation that PSI modulates male-specific somatic or germ-line signals, or a combination of both, that control spermatogenesis. Such signals may be absent or defective in the sterile v16;P[PSIDeltaAB] males (Labourier, 2002).

Because there is almost no postmeiotic transcription during spermatogenesis, RNA-binding proteins involved in nuclear pre-mRNA processing, nucleo-cytoplasmic export, RNA stability, or translation initiation play a crucial role in sperm maturation. PSI is highly expressed in primary spermatocytes and in large mature spermatocytes. It is precisely during this growth and gene expression period that the cells transcribe and process most, if not all, of the gene products needed for the subsequent, major, morphogenetic events of sperm development. An alteration of this critical genetic program in v16;P[PSIDeltaAB] males dramatically affects meiosis and the subsequent spermatid elongation and maturation stages. Lack of the PSI-U1 snRNP association in v16;P[PSIDeltaAB] testes leads to incorrect processing of specific PSI-bound transcripts, such as the hrp40/squid mRNA. squid is required for dorsoventral axis patterning during Drosophila oogenesis, and the different Sqd protein isoforms perform overlapping, but nonequivalent, functions in the localization and translational regulation of specific mRNAs in the female germ line. Sqd may also play an essential role in the male germ line, and a twofold reduction of SqdA expression in the testes may contribute to the v16;P[PSIDeltaAB] male sterility phenotype. In agreement with this model, the absence of the PSI AB motif does not alter the squid splicing pattern in ovaries, and oogenesis proceeds normally in v16;P[PSIDeltaAB] females (Labourier, 2002).

Squid and the apical transport of FTZ mRNA

Drosophila melanogaster pair-rule segmentation gene transcripts localize apically to nuclei in blastoderm embryos. This might occur by asymmetric (vectorial) export from one side of the nucleus or by transport within the cytoplasm. Naked, microinjected Fushi tarazu (Ftz) transcripts do not localize in blastoderm embryos, indicating that cytoplasmic mechanisms alone are insufficient for apical targeting. However, prior exposure of FTZ mRNA to Drosophila or human embryonic nuclear extract leads to rapid, specific, microtubule-dependent transport, arguing against vectorial export. Evidence is presented that FTZ transcript localization involves the Squid (Hrp40) hnRNP protein and that the activity of hnRNP proteins in promoting transcript localization is evolutionarily conserved. It is proposed that cytoplasmic localization machineries recognize transcripts in the context of nuclear partner proteins (Lall, 1999).

Imp associates with Squid and Hrp48 and contributes to localized expression of gurken in the oocyte

Localization and translational control of Drosophila gurken and oskar mRNAs rely on the hnRNP proteins Squid and Hrp48, which are complexed with one another in the ovary. IGF-II mRNA-binding protein (Imp), the Drosophila homolog of proteins acting in localization of mRNAs in other species, is also associated with Squid and Hrp48. Notably, Imp is concentrated at sites of gurken and oskar mRNA localization in the oocyte, and alteration of gurken localization also alters Imp distribution. Imp binds gurken mRNA with high affinity in vitro; thus, the colocalization with gurken mRNA in vivo is likely to be the result of direct binding. Imp mutants support apparently normal regulation of gurken and oskar mRNAs. However, loss of Imp activity partially suppresses a gurken misexpression phenotype, indicating that Imp does act in control of gurken expression but has a largely redundant role that is only revealed when normal gurken expression is perturbed. Overexpression of Imp disrupts localization of gurken mRNA as well as localization and translational regulation of oskar mRNA. The opposing effects of reduced and elevated Imp activity on gurken mRNA expression indicate a role in gurken mRNA regulation (Geng, 2006).

Imp is the Drosophila homolog of a family of proteins that act in posttranscriptional regulation in a variety of animals. One of the founding members of the family, ZBP-1, binds to a localization element in the chicken beta-actin mRNA and appears to direct localization to the leading edge of embryonic fibroblasts. Another founding member, the Xenopus Vg1RBP/VERA protein, binds to signals directing localization of Vg1 and VegT mRNAs to the vegetal pole of the oocyte. Mammalian homologs, the Imp proteins, have been suggested to act in mRNA localization, mRNA stability, and translational regulation. A recent report (Munro, 2006) examined the RNA binding properties of Drosophila Imp protein, focusing specifically on the osk mRNA and its possible regulation by Imp. Although mutation of candidate Imp binding sites in the osk mRNA did block accumulation of Osk protein, loss of Imp activity did not cause a similar defect. This study shows that Imp interacts with Sqd and Hrp48, two proteins that regulate expression of osk and grk mRNAs. Mutation of the Imp gene does not substantially alter grk or osk expression. Nevertheless, the Imp mutant partially suppresses a grk misexpression phenotype, arguing that it does contribute to grk regulation but may act redundantly and does not have an essential role. Consistent with this interpretation, overexpression of Imp interferes with localization of grk mRNA (Geng, 2006).

Deployment of proteins that control patterning in the oocyte relies on coordinated programs of mRNA localization and translational control. Many RNA binding proteins contribute to these programs, and some interact with one another in regulatory RNPs. This study has shown that Imp is associated in an RNA-dependent manner with Sqd and Hrp48 and is thus part of a complex whose other members have clearly established roles in control of grk and osk expression. Imp does not have an essential role in regulation of either grk or osk mRNAs; both mRNAs are expressed with no obvious defects in Imp mutant ovaries. However, loss of Imp activity does partially suppress the grk misexpression defect in fs(1)K10 mutant oocytes, providing strong evidence that Imp contributes to regulation of grk. This view is reinforced by the colocalization of Imp with grk mRNA in vivo. Imp's role must be largely redundant, only becoming detectable when grk expression is perturbed. Overexpression of Imp has a much more dramatic effect, transiently blocking the dorsal localization of grk mRNA and disrupting localization and translational control of osk mRNA (Geng, 2006).

Squid/hnRNP helps Dynein switch from a gurken mRNA transport motor to an ultrastructural static anchor in sponge bodies

In Drosophila oocytes, gurken mRNA localization orientates the TGF-α signal to establish the anteroposterior and dorsoventral axes. This study has elucidated the path and mechanism of gurken mRNA localization by time-lapse cinematography of injected fluorescent transcripts in living oocytes. gurken RNA assembles into particles that move in two distinct steps, both requiring microtubules and cytoplasmic Dynein. gurken particles first move toward the anterior and then turn and move dorsally toward the oocyte nucleus. Evidence is presented suggesting that the two steps of gurken RNA transport occur on distinct arrays of microtubules. Such distinct microtubule networks could provide a general mechanism for one motor to transport different cargos to distinct subcellular destinations (Delanoue, 2007).

This study analyzed the molecular mechanism of grk mRNA transport and anchoring in the Drosophila oocyte using a number of novel methods, combining live cell imaging of oocytes with immunoelectron microscopy to covisualize grk RNA and transacting factors. grk mRNA is transported in particles containing many individual RNA molecules assembled with numerous molecules of Dynein motor components and Squid. Approximately two thirds of transport particles are in close association with MTs and are not consistently associated with membranes, such as ER or vesicles. This supports the idea that grk RNA particles are transported directly by motors on MTs. This notion is strengthened by the fact that the directed movement of the transport particles is disrupted very rapidly when MTs are depolymerized and Dhc, BicD, or Egl function is inhibited. Furthermore, the particles observed moving along MTs in live cell imaging experiments correspond well to the similar-sized grk RNA-rich particles that were visualized by EM. The direct movement of grk RNA particles along MTs is in stark contrast to the transport of yeast ASH1 RNA, which is thought to be cotransported with ER membrane (Delanoue, 2007).

Once delivered to its final destination at the oocyte dorso-anterior corner, many copies of both injected grk RNA and endogenous grk mRNA are anchored in large electron-dense structures previously described as sponge bodies, together with the same components present in the transport particles, including Dynein and Squid. Sponge bodies are distinct in appearance from transport particles and have been previously described in nurse cells and hypothesized to be RNA transport intermediates from the nurse cells to the oocyte. Although Exu-GFP was found in grk anchoring structures, these structures have been identified in the oocyte as functioning in anchoring, rather than transport, and containing components of the Dynein complex Dhc, Egl, and BicD. These data show that the endoplasmic reticulum is not involved in the transport and anchoring of grk mRNA (Delanoue, 2007).

Transport particles and sponge bodies are related to RNA particles (also termed germinal granules, P bodies, and neuronal granules) that display a large spectrum of sizes, composition, and morphology, reflecting several functions in RNA transport, storage, translational control, and processing. Nevertheless, it seems likely that the transport particles that were identified are related to bcd and osk mRNA granules as well as to neuronal RNA granules. The data demonstrate that sponge bodies play key roles in RNA anchoring, but it is not known whether they are also involved in translational control, degradation, and storage of mRNA (Delanoue, 2007).

Dynein is not only present in the sponge bodies but is also required for the static anchoring of grk RNA in the sponge bodies. However, anchoring does not require Egl or BicD, the motor cofactors that are required for RNA transport in the embryo and oocyte. While it is not certain whether Egl and BicD are required for cargo loading, transport initiation, or motor activity itself, the evidence shows that none of these functions are required for anchoring. grk RNA is therefore anchored by a similar mechanism to pair-rule and wingless transcripts in the syncytial blastoderm embryo. In both the embryo and oocyte, it is proposed that when the Dynein motor complex reaches its final destination, the motor becomes a static anchor that no longer depends on the transport activity of the motor (Delanoue, 2007).

Dynein (but not Egl and BicD) is not only a static anchor but is also required for the structural integrity of the grk RNA anchoring structures in the oocyte, the sponge bodies. Their rapid speed of disassembly upon Dynein inhibition (3-5 min) argues that Dynein has a direct role in anchoring and is required to form and maintain the large RNP complexes that constitute the sponge bodies. This evidence rules out that Dynein could indirectly be required for the delivery of anchoring components that are then used in anchoring grk when it is delivered to the sponge bodies. Dynein could also tether the cargo complex directly on the MTs when the transport particles reach their final destination, but the results show that MTs only flank the sponge bodies and are not, as predicted by this model, consistently interdigitated with most of the cargo and motor molecules that are detected in the sponge bodies. This is also consistent with the fact that disassembling MTs does not lead to a change in sponge body structure and only leads to a partial loss of endogenous grk mRNA anchoring (Delanoue, 2007).

In addition to its previously documented role in the second step of grk mRNA transport, a novel function was identified for hnRNP Squid, which plays an essential role in the anchoring of grk RNA at the dorso-anterior corner. Like Dynein, Sqd is also enriched at the site of anchoring upon injection of excess grk RNA. Inactivation of Sqd before transport begins leads to grk transport particles being present at the anterior of the oocyte in permanent anterior flux without anchoring, even for the particles that reach the dorso-anterior corner. Conversely, inactivation of Sqd after grk RNA arrives at the dorso-anterior corner leads to a breakdown of anchoring and the conversion of sponge bodies into anterior transport particles containing grk RNA. This suggests an active role for Sqd in keeping anchoring structures intact, and most likely a role for Sqd in promoting the conversion of transport particles into anchoring structures by facilitating their reorganization into anchoring complexes (Delanoue, 2007).

It is proposed that sponge bodies are assembled at the dorso-anterior corner by delivery of grk mRNA, Dynein motor component and Squid present together in transport particles. First, the same components are present in the transport particles and in the sponge bodies. Second, some transport particles containing endogenous grk mRNA are detected on dorso-anterior MTs. Third, injection of a large excess of grk RNA leads to an increase in the size and number of sponge bodies (Delanoue, 2007).

At the dorso-anterior corner, sponge bodies are maintained by both Dynein and Squid. When the Dynein motor complex reaches its final destination, the motor becomes a static anchor that no longer depends on the transport activity of the motor. Given the size of Dhc and the presence of many putative domains whose function remains elusive but that could play a central role in the switch from motor to anchor, it is proposed that Dhc can associate with many other cellular factors to form a large and immobile anchoring complex. Sqd is known to be involved in translational regulation, and it is proposed that association with this class of factors could help create a large and immobile anchoring complex. A strong link between molecular motors and hnRNPs has already been shown to control the localization of their associated mRNAs. For instance, She2 hnRNP acts as a linker between the Myosin motor complex and its mRNA cargo. Kinesin and hnRNP are present together in RNA-transporting granules in neurons. It is proposed that transport and anchoring of mRNA by molecular motors involve assembly into transport particles followed by reconfiguration of the same components into large electron-dense anchoring complexes at the final destination. Future work will establish how widely this model can be applied. It will also be interesting to determine whether the specificity of transport and anchoring of other RNA cargos in the Drosophila oocyte and embryo is also established by distinct combinations of RNA-binding proteins that influence the function of molecular motors. Time will tell what proportion of mRNA is anchored like pair-rule and grk transcripts by static functions of molecular motors, as opposed to other possible mechanisms of anchoring (Delanoue, 2007).

squid: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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