BIOLOGICAL OVERVIEW

Ypsilon schachtel (Yps), a Drosophila Y-box protein ('schachtel' is German for 'box'), is a subunit of a cytoplasmic RNA-protein complex hypothesized to regulate subcellular RNA localization in Drosophila ovaries (Wilhelm, 2000). Yps binds directly to Exuperantia (Exu) and colocalizes with Exu in both the oocyte and nurse cells of the Drosophila egg chamber. The Exu-Yps complex contains OSK mRNA (Wilhelm, 2000). yps interacts genetically with oo18 RNA-binding protein (orb), a positive regulator of Oskar mRNA localization and translation. The nature of the genetic interaction indicates that yps acts antagonistically to orb. Orb protein is physically associated with both the Yps and Exu proteins, and this interaction is mediated by RNA. A model is proposed wherein Yps and Orb bind competitively to Oskar mRNA with opposite effects on translation and RNA localization (Mansfield, 2002).

Y-box proteins are present in a diverse set of species, including worms, flies, frogs, mice and humans. All members of this family of proteins contain a well-defined nucleic acid binding domain, the cold shock domain. In vertebrate germ cells, Y-box proteins are major cellular components. Biochemical evidence indicates that some of these proteins bind mRNA and regulate translation. For example, FRGY2 in Xenopus, and MSY2 and MSY4 in the mouse, repress translation of mRNAs in oocytes and spermatocytes. In somatic cells, Y-box proteins are also implicated in translational regulation. The p50/YB-1 proteins are highly conserved across vertebrate species and can enhance or mask translation in a concentration-dependent manner. The mechanism by which such translational masking and enhancement is accomplished is not well understood, although it appears to involve modulation of mRNA secondary structure (Mansfield, 2002).

Prior to this work, only one member of the Y-box family had been genetically characterized, the C. elegans heterochronic gene lin-28, which functions in the development of somatic cell lineages (Moss, 1997). Targets of Lin-28 activity have not yet been reported, and while it is predicted to be an RNA-binding protein, its mode of action is not known. The null allele of yps, yps^JM2, produced by excising a P-element, contains an internal deletion in the gene that removes the start codon and the N-terminal half of the protein, including the entire cold shock domain. Several lines of evidence indicate that OSK mRNA, which encodes a primary organizer of the germ plasm, is a target of Yps' activity. (1) OSK mRNA coimmunoprecipitates with both Yps and Exu proteins from ovary extracts (Wilhelm, 2000). (2) OSK mRNA colocalizes with Yps and Exu throughout oogenesis. (3) There is a robust genetic interaction between yps and orb: keeping in mind orb's known regulation of OSK mRNA translation and localization, the yps null allele rescues orb-associated defects in OSK mRNA localization and translation (Mansfield, 2002).

In intermediate allelic combinations of orb, OSK mRNA fails to localize to the posterior pole of the oocyte, and Osk protein is not translated. The localization and translation of OSK mRNA is subject to a complex autoregulatory loop, whereby OSK mRNA must first be localized to the posterior pole of the oocyte to be translated, and subsequently Osk protein is required to maintain the localization of its own mRNA. Because the localization and translation processes are so entwined, it can be difficult to establish which process a regulatory factor affects. In the case of Orb, however, evidence suggests that its primary function may be translational regulation of OSK mRNA. In Xenopus, CPEB, which is virtually identical to Orb in its RNA-binding domain, regulates translation of stored maternal mRNAs by binding a U-rich region of 3'UTRs (the cytoplasmic polyadenylation element) and promoting cytoplasmic polyadenylation. The role of OSK mRNA's poly(A) tail in translation is controversial. Results from in vitro systems developed to study translation in Drosophila ovaries suggest that the length of OSK's poly(A) tail is not critical for regulating its translation. However, in vivo studies of OSK mRNA suggest that poly(A) tail length does affect its translation. These latter results indicate that polyadenylation of the OSK transcript is dependent on the function of orb, as is accumulation of Osk protein, suggesting that Orb serves a similar function to that of CPEB. In addition, Orb binds specifically to the OSK 3'UTR. Given this evidence, it appears that Orb may function as a translational enhancer of Osk, although a direct role in OSK mRNA localization cannot be ruled out (Mansfield, 2002 and references therein).

The orb genotypes that are rescued in double mutant combinations with yps^JM2 all include the orb^mel mutation, a hypomorphic allele that produces some functional Orb protein. In contrast, females homozygous for a null allele (orb^F343) or a strong allele (orb^F303) show no rescue by the yps^JM2 mutation. These results indicate that rescue by yps^JM2 requires the presence of some functional Orb protein, and that Yps may normally act antagonistically to Orb. In the presence of Yps, the low amount of functional Orb protein present in orb^mel mutants is not capable of promoting normal OSK mRNA localization and translation, whereas in the absence of Yps, the reduced Orb protein is sufficient (Mansfield, 2002).

The data indicate that yps is unlikely to regulate the expression or localization of Orb protein itself. (1) The distribution and levels of Orb produced by hypomorphic orb alleles are not altered in a yps^JM2 background. In addition, genetic analysis of yps, orb double mutants, shows that yps^JM2 specifically rescues defects in OSK mRNA localization and translation, but not orb-associated defects in dorsoventral chorion patterning or grk mRNA localization. Taken together, these results indicate a specific effect of yps on orb's function in localizing and/or translating OSK mRNA (Mansfield, 2002).

Previous work has shown that, in the minority of orb^mel egg chambers in which Osk protein is detectable, Orb protein can be detected at the posterior pole as well. This correlation has been interpreted as evidence of a requirement for Orb for the on-site expression of Osk. When ovaries are doubly mutant for yps and orb, this correlation disappears. While Orb can rarely be detected at the posterior pole of the oocyte in yps;orb mutants, Osk protein is frequently present even in the absence of detectable Orb. However, loss of Yps cannot eliminate the requirement for Orb in Osk expression. It is possible that, in the absence of Yps, a very low concentration of Orb, which is undetectable by immunocytochemistry, is sufficient to localize or enhance the translation of OSK mRNA at the posterior pole. Alternatively, in the absence of Yps, the function of Orb might be accomplished at regions other than the posterior, since in yps;orb double mutants Orb protein is present throughout the oocyte (Mansfield, 2002).

Although OSK mRNA translation is significantly rescued in yps;orb ovaries, the amount of Osk present at the posterior appears reduced compared to wild type. In addition, Osk is not reliably detected in yps;orb egg chambers until stage 10. In wild-type ovaries, however, Osk can be detected in stage-9 oocytes, and sometimes as early as stage 8. It is thought that the temporal delay in detecting Osk is due simply to a reduction in Osk expression in yps;orb egg chambers during all stages of oogenesis, such that accumulation of the protein to detectable levels does not occur until stage 10. It is also hypothesized that, due to this reduction in the accumulation of Osk protein in yps;orb ovaries, OSK mRNA localization is not efficiently maintained. In late stage 9, 66% of yps;orb oocytes displayed localized OSK mRNA, while in stage 10 the percentage falls to 45%. This number closely parallels the percentage of yps;orb stage 10 oocytes with detectable Osk protein (43%) and the number of eggs (40%) that hatched from mutant mothers (Mansfield, 2002).

Biochemically an association between Yps and Orb has been detected. Orb protein coimmunoprecipitates with Yps. This association is mediated by RNA, since their coimmunoprecipitation is RNAse-sensitive. Similarly, Orb coimmunoprecipitates with Exu, in an RNA-dependent manner. Exu and Yps also coimmunoprecipitate, but independently of RNA, and bind each other in vitro, indicating that their interaction is probably direct (Wilhelm, 2000). Despite their direct association, Yps is localized normally in exu null ovaries, and Exu protein is localized normally in yps null ovaries. Thus Yps and Exu appear to be recruited independently to this ovarian complex. Do the associations detected by immunoprecipitation reflect biologically significant interactions that occur in vivo? Several other lines of evidence suggest that these proteins interact in vivo, and that OSK mRNA is part of this complex: (1) all three proteins, and OSK mRNA, colocalize throughout oogenesis; (2) OSK mRNA associates with both Exu and Yps (Wilhelm, 2000), and Orb binds directly to OSK mRNA; (3) the genetic results presented in this work are strong evidence for a biologically significant interaction of Yps and Orb in Drosophila ovaries (Mansfield, 2002).

Doubly mutant yps^JM2orb^mel/yps^JM2orb^F303 ovaries display a novel phenotype, not observed in yps^JM2 or orb^mel/orb^F303 females: a small proportion (5%) of mid- and late-stage egg chambers are bipolar. Strong allelic combinations of orb also generate a high proportion of egg chambers with the oocyte mispositioned, but these egg chambers arrest oogenesis before budding from the germarium, or shortly thereafter. The low frequency of late-stage bipolar egg chambers observed in yps^JM2orb^mel/yps^JM2orb^F303 females may result from partial rescue of egg chambers that would normally have arrested at very early stages in orb^mel /orb^F303 ovaries, with a phenotype similar to orb^F303/orb^F303 egg chambers. Alternatively, this may be a novel phenotype resulting from the additive loss of both yps and orb. In either case, this phenotype suggests an earlier, as yet uncharacterized function of yps. In support of this idea, yps is expressed in the germarium (Mansfield, 2002).

In addition to its expression in female germ cells, yps is also expressed in the testes and in somatic cells (Thieringer, 1997; Wilhelm, 2000). It is therefore unlikely that OSK mRNA is the only target of Yps. In fact, most Y-box proteins are general factors that regulate the translation of large classes of mRNAs. FRGY2, for example, is thought to be a general masking factor for stored maternal mRNAs in the oocyte. However, there is no evidence to suggest that Yps serves such a general role in translational repression. The fact that Yps is localized to specific subcellular sites in the nurse cells and the oocyte argues against it being a general regulator of translation. The hypothesis is favored that Yps is specific to a limited number of target mRNAs. Perhaps Exu, which has been shown to coimmunoprecipitate with both Yps protein and OSK mRNA, or another protein in the complex, confers sequence specificity on Yps' RNA-binding activity (Mansfield, 2002).

An interesting possibility is suggested by studies of the p50/YB1 Y-box proteins. p50/YB1 is nearly completely conserved across vertebrate species, including human, mouse, rabbit and chick, and is expressed in both germline and somatic cells. The most abundant protein in mRNP particles in a variety of cells types, p50/YB1, has been shown to act as a translational repressor at high concentrations and a translational enhancer at low concentrations. Recent work has shown that, at low concentrations (i.e., when mRNAs are unmasked) p50/YB1 also binds actin, and at higher p50/YB1: mRNA ratios (conditions under which translation is repressed) p50/YB1 and actin do not bind (Ruzanov, 1999). Increasing YB-1 mRNA binding activity or concentration in cells activates a general default pathway for mRNA stabilization (Evdokimova, 2001). YB-1 interaction with actin suggests a mechanism by which unmasked mRNAs may be anchored to regions of the cytoplasm. This is particularly intriguing in the light of evidence that OSK mRNA is anchored by an actin-dependent mechanism to the posterior pole of the oocyte, where it is translated. While Yps cannot be an essential component of a posterior RNA anchor, it may play a role in the association of OSK mRNA with the actin cytoskeleton, which may in turn affect translation (Mansfield, 2002).

One model supported by the data is that Yps and Orb both bind to OSK mRNA, and have opposite effects on translation: Yps represses, and Orb activates translation. Immunoprecipitation experiments show that both proteins are present in an RNP complex and that their association is mediated by RNA, suggesting that both proteins simultaneously bind a common RNA target. This target is likely to be OSK mRNA. OSK mRNA is a member of this RNP complex (Wilhelm, 2000). Orb is known to bind OSK mRNA, and the genetic results show that a yps loss-of-function mutation suppresses the defects in OSK mRNA localization and translation associated with reduced function orb alleles. Yps could prevent translation by preventing Orb from promoting cytoplasmic polyadenylation. At the posterior of the oocyte, where Orb and Yps both concentrate during mid-oogenesis, and where OSK mRNA is localized and translated, concentration differences between the two proteins could push the complex from being a negative to a positive regulator of translation. Additional factors at the posterior could also interact with either Orb or Yps to modify their functions, as might association with the actin cytoskeleton. This model accounts for why the yps mutation cannot eliminate the requirement for Orb, but can reduce the amount of Orb required for sufficient OSK mRNA translation. In the rescued genotypes, there may be enough Orb at the oocyte posterior to allow for on-site cytoplasmic polyadenylation of OSK mRNA, in the absence of negative regulation by Yps. It is also possible that, in the absence of Yps, Orb can stimulate polyadenylation of OSK mRNA before it becomes localized, although it remains subject to translational repression by other factors, such as Apontic and Bruno, until it reaches the posterior pole. Future studies will test this model by determining if Yps and Orb bind competitively to OSK mRNA, and if so, how their combined binding affects the translation of OSK mRNA, and its polyadenylation state. These studies should contribute not only to an understanding of localization-dependent mRNA translation in Drosophila, but also to a better understanding of the biological roles of the widespread family of Y-box proteins (Mansfield, 2002).

GENE STRUCTURE

Northern blot analysis, using a yps cDNA to probe poly(A+)RNA isolated from hand-dissected ovaries, has revealed the presence of two species of transcripts: a broad mRNA band migrating at approximately 1.7 kb, which may contain several transcripts of similar size, and a minor 2.3 kb transcript. Alignment of previously published yps cDNA sequences (Thieringer, 1997; Wilhelm, 2000) with genomic DNA reveals the possibility of an alternate fourth exon, which could account for the size difference of approximately 600 bp between the larger and smaller transcripts. In addition, this predicted exon contains multiple consensus polyadenylation signals, which could produce the broad 1.7 kb band observed on Northern blots. If used, this exon would replace the last 15 amino acids of the known protein sequence with 23 alternative amino acids (Mansfield, 2002).

PROTEIN STRUCTURE

Amino Acids - 359

Structural Domains

Yps, while containing a conserved cold shock domain (CSD), is novel in that it completely lacks the alternating acidic and basic regions found in the C-terminus of the other vertebrate eukaryotic Y-box proteins. The CSD of yps was purified and gel-shift analysis shows that this domain can interact with RNA. It is predicted that Yps would be an RNA-binding protein due to these results and the motifs which have been identified within the amino acid sequence (Thieringer, 1997).

The Yps protein has been aligned with the CSD of other known eukaryotic Y-box proteins, along with CspA from E. coli. Identity within this region is 89% compared to the human YB-1 protein and 47% compared to CspA. The RNP1 RNA binding motif encompasses the sequence GYGF and is conserved among all the eukaryotic proteins. In addition, the RNP-2 like motif VFV is also conserved among all the proteins, with the exception of C. elegans, which contains the sequence LFV (Thieringer, 1997).

Most Y-box proteins do not have high sequence homology with each other when comparing the C-terminal domains. It has been proposed that this domain has eight regions of alternating acidic and basic residues, each about 30 amino acid residues in length. The eight alternating regions are clearly visible in the vertebrate Y-box proteins. However, these regions are absent in the Drosophila yps protein, and in the Y-box protein from Aplysia californica. In both of these cases there is only one acidic region within an entirely basic tail domain. The similarity between Aplysia and Drosophila would suggest that the invertebrate proteins may make up a different class of Y-box proteins than the vertebrate members. Interestingly, the C. elegans gene lin-28 encodes a protein with a much shorter C-terminal tail compared to the other eukaryotic Y-box proteins, and also does not have alternating acidic and basic regions. In addition, a plant Y-box protein, GRP, does not have the alternating acidic and basic residues at the C-terminus, however this protein is much smaller than other Y-box proteins and is glycine-rich (Thieringer, 1997 and references therein).

One characteristic which is common to all the vertebrate Y-box proteins is a high arginine content in the C-terminal tail of these proteins. This is also true for the yps protein. The RGG box which is known as an RNA binding domain, is defined as closely spaced Arg-Gly-Gly (RGG) repeats. The minimal number of RGG repeats required for RNA binding is not known, however the yps protein contains four repeats located in the tail region between amino acid residues 204 and 276 (Thieringer, 1997).

The CSD of the Yps protein was successfully purified by the use of ion-exchange chromatography to over 95% purity. This purified protein was then used for studies of its nucleic acid binding characteristics. The probe used to measure the RNA binding capability of the CSD of yps was a single-stranded 38mer; the CSD of yps is able to bind to this RNA probe, when added at concentrations of 8 and 12 microM. When protein is added to the probe at concentrations of 2 microM, no gel shift is seen, and at 4 microM only a partial gel shift is observed. 120 pmol of the CSD was required to bind 25 fmol of the RNA probe (Thieringer, 1997).

To identify the 57-kD Exu-associated protein, three tryptic peptides were microsequenced from the purified protein. The sequence from two of the three peptides matched a previously identified protein, the product of the yps gene. Yps is a member of the cold shock family of RNA-binding domain proteins and was identified as part of a degenerate PCR screen to identify cold shock domain containing genes from Drosophila melanogaster (Thieringer, 1997). However, the third peptide only matched the Yps sequence in a reading frame other than the published open reading frame. To rule out the possibility that yps expression is subject to ribosomal frameshifting or RNA editing, six independently sequenced yps ESTs were obtained. Sequence analysis revealed that the original yps sequence contained several sequencing errors and that the correct open reading frame contains all three microsequenced peptides. The cold shock domain of Yps shows extensive sequence identity to other cold shock domain proteins. This domain has been shown in several studies to bind RNA, although its ability to recognize specific substrates remains uncertain. Beyond the cold shock domain, Yps exhibits no significant homology to any other protein except YB-1, a cold shock domain protein from Drosophila silvesteris (AAC06034). Since the YB-1 protein is 70% identical to Yps across the entire length of the protein, it is likely to be a true ortholog of Yps. No function was assigned to either YB-1 or Yps in these prior studies (Wilhelm, 2000).

date revised: 25 February 2002

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