Vegetal Cortex Of Xenopus Oocytes Research Articles

A novel role for Celf1 in vegetal RNA localization during Xenopus oogenesis

The localization of certain mRNAs to the vegetal cortex of Xenopus oocytes is of crucial importance for germ cell development and early embryonic patterning. Vegetal RNA localization is mediated by cis-acting RNA localization elements (LE). Several proteins assemble on the RNA LE and direct transport to the vegetal cortex. Although a number of localization RNP components have been identified, their full composition is unknown. In an RNA affinity purification approach, using the dead end 1 (dnd1) RNA LE, we identified Xenopus Celf1 as a novel component of vegetal localization RNP complexes. Celf1 is part of an RNP complex together with known vegetal localization factors and shows specific interactions with LEs from several but not all vegetally localizing RNAs. Immunostaining experiments reveal co-localization of Celf1 with vegetally localizing RNA and with known localization factors. Inhibition of Celf1 protein binding by localization element mutagenesis as well as Celf1 overexpression interfere with vegetal RNA localization. These results argue for a role of Celf1 in vegetal RNA localization during Xenopus oogenesis.

Directional Transport Is Mediated by a Dynein-Dependent Step in an RNA Localization Pathway

Author SummaryLike traffic on highways, molecular cargos are transported within cells on tracks that are collectively referred to as cytoskeletal networks. RNA molecules are one such cargo, and in many species, the localization of RNAs in egg cells or oocytes is essential for establishing the first asymmetries that are necessary for proper embryo development. RNAs can be actively transported by molecular motors that move cargos along the cytoskeletal tracks, but how such motors are capable of directing cargos to specific destinations within the cell is not yet known. Here we show that two motors, dynein and kinesin—known to carry out transport in opposite directions—are both directly involved in RNA localization in frog oocytes. To understand how these motors can promote directional cargo transport, we developed a system to monitor RNA transport in live oocytes. We find that the motor acting first in the pathway, dynein, is responsible for unidirectional transport. Bidirectional transport, mediated by kinesin, occurs subsequently on cytoskeletal tracks of opposing polarity near the RNA's final destination. Our results suggest a new model for directional transport comprising an initial directional cue that dominates over a later nondirectional step, acting to refine the ultimate cargo distribution.

Structural messenger RNA contains cytokeratin polymerization and depolymerization signals

We have previously shown that VegT mRNA plays a structural (translation-independent) role in the organization of the cytokeratin cytoskeleton in Xenopus oocytes. The depletion of VegT mRNA causes the fragmentation of the cytokeratin network in the vegetal cortex of Xenopus oocytes. This effect can be rescued by the injection of synthetic VegT RNA into the oocyte. Here, we show that the structural function of VegT mRNA in Xenopus oocyte depends on its combinatory signals for the induction or facilitation and for the maintenance of the depolymerization vs. polymerization status of cytokeratin filaments and that the 300-nucleotide fragment of VegT RNA isolated from the context of the entire molecule induces and maintains the depolymerization of cytokeratin filaments when injected into Xenopus oocytes. A computational analysis of three homologous Xenopus VegT mRNAs has revealed the presence, within this 300-nucleotide region, of a conserved base-pairing (hairpin) configuration that might function in RNA/protein interactions.

Functional dissection of the RNA signal sequence responsible for vegetal localization of XGrip2.1 mRNA in Xenopus oocytes

Grip2.1 is a conserved PDZ-domain protein with a function in the context of primordial germ cell development and migration in Xenopus embryos. Its mRNA is maternally supplied and found to be associated with the germ plasm, located at the tip of the vegetal cortex in Xenopus oocytes. Here, we demonstrate that the 3’-UTR of XGrip2.1 contains a 211 nucleotide RNA signal sequence that promotes localization to the mitochondrial cloud via the early localization pathway upon injection into stage I oocytes. The same element is also capable of using the late transport pathway if injected into stage III/IV oocytes. In vitro protein interaction studies reveal binding to ElrA/B, Vg1RBP and VgRBP60, proteins that have previously been associated with the vegetal localization machinery. Mutational interference with Vg1RBP and VgRBP60 binding severely reduces early and late localization activity. Selective interference with Vg1RBP binding significantly reduces late localization while having only a mild effect on localization to the mitochondrial cloud, indicating that the signal sequences and protein machinery required for early and late pathway localization though overlapping are not identical.

Interaction of 42Sp50 with the vegetal RNA localization machinery in Xenopus laevis oocytes

Localization of a specific subset of maternal mRNAs to the vegetal cortex of Xenopus oocytes is important for the regulation of germ layer formation and germ cell development. It is driven by vegetal localization complexes that are formed with the corresponding signal sequences in the untranslated regions of the mRNAs and with a number of different so-called localization proteins. In the context of the present study, we incorporated tagged variants of the known localization protein Vg1RBP into vegetal localization complexes by means of oocyte microinjection. Immunoprecipitation of the corresponding RNPs allowed for the identification of novel Vg1RBP-associated proteins, such as the embryonic poly(A) binding protein, the Y-box RNA-packaging protein 2B and the oocyte-specific version of the elongation factor 1α (42Sp50). Incorporation of 42Sp50 into localization RNPs could be confirmed by co-immunoprecipitation of Vg1RBP and Staufen1 with myc-tagged 42Sp50. Furthermore, myc-42Sp50 was found to co-sediment with the same two proteins in large, RNAse-sensitive complexes, as well as to associate specifically with several vegetally localizing mRNAs but not with nonlocalized control RNAs. Finally, oocyte microinjection experiments reveal that 42Sp50 is a protein that shuttles between the nucleus and cytoplasm. Taken together, these observations provide evidence for a novel function of 42Sp50 in the context of vegetal mRNA transport in Xenopus oocytes.

Vg1 RNA localization in oocytes in the absence of xVICKZ3 RNA-binding activity

Vg 1 RNA becomes localized at the vegetal cortex of Xenopus oocytes in a process requiring both intact microtubules (MT) and microfilaments. This localization occurs during a narrow window of oogenesis, when a number of RNA-binding proteins associate with the RNA. xVICKZ3 (Vg1 RBP/Vera), the first Vg1 RNA-binding protein identified, helps mediate the association of Vg1 RNA with MT and is co-localized with the RNA at the vegetal cortex. Given the complexity of the Vg1 RNA ribonucleoprotein (RNP) complex, it has remained unclear how xVICKZ3 functions in Vg1 RNA localization. Here, we have taken a closer look at the process of xVICKZ3 localization in oocytes. We have made use of deletion constructs to perform a structure–function analysis of xVICKZ3. The ability of xVICKZ3–GFP constructs to vegetally localize correlates with their association to MT but not with Vg1 RNA-binding ability. We find that when the ability of xVICKZ3 to bind Vg1 RNA is inhibited by the injection of a construct that dominantly inhibits RNA binding, both the construct and Vg1 RNA still localize, apparently through their continued association with a Vg1 RNA-containing RNP complex. These results emphasize the importance of protein–protein interactions in both xVICKZ3 and Vg1 RNA localization.

Organization of cytokeratin cytoskeleton and germ plasm in the vegetal cortex of Xenopus laevis oocytes depends on coding and non-coding RNAs: Three-dimensional and ultrastructural analysis

Recent studies discovered a novel structural role of RNA in maintaining the integrity of the mitotic spindle and cellular cytoskeleton. In Xenopus laevis, non-coding Xlsirts and coding VegT RNAs play a structural role in anchoring localized RNAs, maintaining the organization of the cytokeratin cytoskeleton and germinal granules in the oocyte vegetal cortex and in subsequent development of the germline in the embryo. We studied the ultrastructural effects of antisense oligonucleotide driven ablation of Xlsirts and VegT RNAs on the organization of the cytokeratin, germ plasm and other components of the vegetal cortex. We developed a novel method to immunolabel and visualize cytokeratin at the electron microscopy level, which allowed us to reconstruct the ultrastructural organization of the cytokeratin network relative to the components of the vegetal cortex in Xenopus oocytes. The removal of Xlsirts and VegT RNAs not only disrupts the cytokeratin cytoskeleton but also has a profound transcript-specific effect on the anchoring and distribution of germ plasm islands and their germinal granules and the arrangement of yolk platelets within the vegetal cortex. We suggest that the cytokeratin cytoskeleton plays a role in anchoring of germ plasm islands within the vegetal cortex and germinal granules within the germ plasm islands.

Delivery of germinal granules and localized RNAs via the messenger transport organizer pathway to the vegetal cortex of Xenopus oocytes occurs through directional expansion of the mitochondrial cloud

During Xenopus oogenesis, the message transport organizer (METRO) pathway delivers germinal granules and localized RNAs to the vegetal cortex of the oocyte via the mitochondrial cloud (Balbiani body). According to the traditional model, the mitochondrial cloud is thought to break up at the onset of vitellogenesis and the germinal granules and METRO-localized RNAs are transported within the mitochondrial cloud fragments to the vegetal cortex of the oocyte. We used light and electron microscopy in situ hybridization and three-dimensional reconstruction to show that germinal granules and METRO-localized RNAs are delivered to the oocyte cortex before the onset of mitochondrial cloud fragmentation and that the delivery involves accumulation of localized RNAs and aggregation of germinal granules at the vegetal tip of the mitochondrial cloud and subsequent internal expansion of the mitochondrial cloud between its animal (nuclear) and vegetal tips, which drives the germinal granules and METRO-localized RNAs toward the vegetal cortex. Thus the fragmentation of the cloud that occurs later in oogenesis is irrelevant to the movement of METRO-localized RNAs and germinal granules. On the basis of these findings, we propose here a revised model of germinal granule and localized RNAs delivery to the oocyte vegetal cortex via the METRO pathway.

Evidence for overlapping, but not identical, protein machineries operating in vegetal RNA localization along early and late pathways in Xenopus oocytes.

RNAs that localize to the vegetal cortex of Xenopus oocytes are involved in early embryonic patterning and cell fate specification. Two mechanistically distinct pathways lead to RNA enrichment at the vegetal cortex: the early and the late. While several candidate proteins that seem to operate in the late localization pathway have been identified, proteins involved in the early pathway remain to be identified. In this study, we report on the isolation of a novel vegetally localized RNA in Xenopus oocytes that makes use of the early pathway and encodes a protein with a conserved but functionally uncharacterized NIF-motif. The localization signal of XNIF was mapped to a 300-nucleotide region in the 5'-UTR, which is able to mediate both accumulation to the mitochondrial cloud in stage I oocytes, as well as vegetal transport in later stage oocytes. The XNIF-LE contains 16 copies of the previously defined CAC-containing signal motifs for RNA localization. A critical number of such repeats seems to be required for accumulation in the mitochondrial cloud along the early pathway, but additional repeats seem to be required for localization along the late pathway. Cross-linking experiments identify two novel proteins of 62 and 64 kDa that interact with the XNIF-LE but not with the Vg1-LE that operates in the late pathway. Conversely, at least two of the previously identified VgRBPs, Vg1RBP1 and Prrp, also bind to the XNIF-LE. Thus, overlapping, but not identical, protein machineries mediate vegetal RNA localization along early and late pathways in Xenopus oocytes.

A Ubiquitous and Conserved Signal for RNA Localization in Chordates

During oogenesis in Xenopus laevis, several RNAs that localize to the vegetal cortex via one of three temporally defined pathways have been identified [1–4]. Although individual mRNAs utilize only one pathway, there is functional overlap [5] and apparent continuity [6] between them, suggesting that common cis-acting sequences may exist. Because previous work with the Vg1 mRNA revealed that short nontandem repeats are important for localization [7, 8], we developed a new computer program, called REPFIND, to expedite the identification of repeated motifs in other localized RNAs. Here we show that clusters of short CAC-containing motifs characterize the localization elements (LEs) of virtually all mRNAs localized to the vegetal cortex of Xenopus oocytes. A search for this signal in GenBank [9] resulted in the identification of new localized mRNAs, demonstrating the applicability of REPFIND to predict localized RNAs. CAC-rich LEs are also found in ascidians and other vertebrates, indicating that these cis regulatory elements are conserved in chordates. Interestingly, biochemical evidence shows that distinct CAC-containing motifs have different functions in the localization process. Thus, clusters of CAC-containing motifs are a ubiquitous signal for RNA localization and can signal localization in a variety of pathways through slight variations in sequence composition.

A Consensus RNA Signal That Directs Germ Layer Determinants to the Vegetal Cortex of Xenopus Oocytes

RNA localization is an important mechanism for generating cellular diversity and polarity in the early embryo. In Xenopus, the correct localization of the RNA encoding the T-box transcription factor VegT is essential for the correct spatial organization and identity of endoderm and mesoderm. Although localization signals in the 3′ UTR have been identified for many localized RNAs, insight into what constitutes an RNA localization signal remains elusive. To investigate possible common features between signals that direct different RNAs to the same subcellular region, we carried out a detailed analysis of the uncharacterized VegT RNA localization signal and compared it with the well-studied Vg1 localization signal. Both RNAs localize to the vegetal cortex during the same period of oogenesis. Our results suggest a common RNA localization signal at the level of clustered redundant protein-binding motifs and trans-acting factors. We propose that what characterizes RNA localization signals in general is not the nucleotide sequence or secondary structure per se, but the critical clustering of specific redundant protein-binding motifs.

A proline-rich protein binds to the localization element of Xenopus Vg1 mRNA and to ligands involved in actin polymerization.

A 340 nucleotide element within the 3' untranslated region of Vg1 mRNA determines its localization to the vegetal cortex of Xenopus oocytes. To identify protein factors that bind to this region, we screened a cDNA expression library with an RNA probe containing this sequence. Five independent isolates encoded a protein (designated Prrp for proline-rich RNA binding protein) having two RNP domains followed by multiple polyproline segments. Prrp and Vg1 mRNAs are co-localized to the vegetal cortex of stage IV oocytes, substantiating an interaction between the two in vivo. Prrp also associates with VegT mRNA, which like Vg1 mRNA uses the late localization pathway, but not with Xcat-2 or Xwnt-11 mRNAs, which use the early pathway. The proline-rich domain of Prrp interacts with profilin, a protein that promotes actin polymerization. Prrp can also associate with the EVH1 domain of Mena, another microfilament-associated protein. Since the anchoring of Vg1 mRNA to the vegetal cortex is actin dependent, one function of Prrp may be to facilitate local actin polymerization, representing a novel function for an RNA binding protein.

Fatvg encodes a new localized RNA that uses a 25-nucleotide element (FVLE1) to localize to the vegetal cortex of Xenopus oocytes.

Vegetally localized transcripts have been implicated in a number of important biological functions, including cell fate determination and embryonic patterning. We have isolated a cDNA, fatvg, which encodes a localized maternal transcript that exhibits a localization pattern reminiscent of Vg1 mRNA. fatvg is the homologue of a mammalian gene expressed in adipose tissues. The fatvg transcript, unlike Vg1 which localizes strictly through the Late pathway, also associates with the mitochondrial cloud that is characteristic of the METRO or Early pathway. This suggests that fatvg mRNA may utilize both the METRO and Late pathways to localize to the vegetal cortex during oogenesis. We have dissected the cis-acting localization elements of fatvg mRNA and compared these elements with Vg1 mRNA. Our results indicate that, like most localized RNAs, in a variety of systems, transcripts of fatvg contain localization elements in the 3'UTR. The 3'UTR of fatvg mRNA contains multiple elements that are able to function independently; however, it functions most efficiently when all of the elements are present. We have defined a short 25-nucleotide element that can direct vegetal localization as a single copy. This element differs in sequence from previously described Vg1 localization elements, suggesting that different localization elements are involved in the localization of RNAs through the Late pathway.

Vg1 RBP intracellular distribution and evolutionarily conserved expression at multiple stages during development

We have analyzed the expression and intracellular distribution, during oogenesis and embryogenesis, of Vg1 RBP, a protein implicated in the intracellular localization of Vg1 mRNA to the vegetal cortex of Xenopus oocytes. Vg1 RBP (protein) colocalizes with Vg1 RNA at all stages of oogenesis. Vg1 RBP RNA, however, localizes to the animal pole during late oogenesis, and remains in the animal blastomeres and ectodermal precursors until its zygotic transcription is activated, around stage 12. Vg1 RBP mRNA then becomes expressed throughout the neural epithelium. Vg1 RBP mRNA expression is also detected in what appears to be neural crest cells undergoing delamination and lateral migration. By tailbud stages, Vg1 RBP expression is present in the branchial arches, otic vesicle, pronephros, and along the neural tube. To examine the expression pattern in different species, we cloned the zebrafish homolog of Vg1 RBP by using a highly homologous EST clone to screen an embryonic cDNA library. In situ hybridization reveals that Vg1 RBP RNA localizes early in oogenesis to the animal pole. Although Vg1 RBP RNA is detected in all blastomeres of the early embryo, the expression pattern in the one day old zebrafish embryo is almost identical to that of the equivalent stage Xenopus embryo. These results indicate that the zygotic expression pattern is similar in frogs and fish, and that there is a conserved zygotic expression of Vg1 RBP distinct from its expression in the oocyte.

RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization.

Vg1 mRNA translocation to the vegetal cortex of Xenopus oocytes requires intact microtubules, and a 3' UTR cis-acting element (termed VLE), which also mediates sequence-specific binding of several proteins. One protein, the 69-kD Vg1 RBP, associates Vg1 RNA to microtubules in vitro. Here we show that Vg1 RBP-binding sites correlate with vegetal localization. Purification and cloning of Vg1 RBP revealed five RNA-binding motifs: four KH and one RRM domains. Surprisingly, Vg1 RBP is highly homologous to the zipcode binding protein implicated in the microfilament-mediated localization of beta actin mRNA in fibroblasts. These data support Vg1 RBP's direct role in vegetal localization and suggest the existence of a general, evolutionarily conserved mechanism for mRNA targeting.

Apparent continuity between the messenger transport organizer and late RNA localization pathways during oogenesis in Xenopus

The localization of RNAs at the vegetal cortex in Xenopus oocytes is a complex process, involving at least two different pathways. The early, or messenger transport organizer (METRO), pathway, localizes RNAs such as Xlsirts, Xcat2 and Xwnt11 during stages 1 and 2 of oogenesis, while the late pathway localizes RNAs such as Vg1 during stages 2–4. We demonstrate that the onset of Vg1 localization is characterized by its microtubule-independent binding to a subdomain of the endoplasmic reticulum (ER). The formation of this unique ER structure is intimately associated with the movement of the mitochondrial cloud toward the vegetal cortex. In addition, we demonstrate that the mitochondrial cloud contains a γ-tubulin-positive structure that may function as a microtubule organizing center for establishing microtubule tracks for Vg1 localization. These data, support, although they do not prove, a model in which the development of the late pathway machinery relies upon the prior functioning of the early pathway.

Two copies of a subelement from the Vg1 RNA localization sequence are sufficient to direct vegetal localization in Xenopus oocytes.

Localization of mRNA has emerged as a fundamental mechanism for generating polarity during development. In vertebrates, one example of this phenomenon is Vg1 RNA, which is localized to the vegetal cortex of Xenopus oocytes. Vegetal localization of Vg1 RNA is directed by a 340-nt sequence element contained within its 3' untranslated region. To investigate how such cis-acting elements function in the localization process, we have undertaken a detailed analysis of the precise sequence requirements for vegetal localization within the 340-nt localization element. We present evidence for considerable redundancy within the localization element and demonstrate that critical sequences lie at the ends of the element. Importantly, we show that a subelement from the 5' end of the Vg1 localization element is, when duplicated, sufficient to direct vegetal localization. We suggest that the Vg1 localization element is composed of smaller, redundant sequence motifs and identify one such 6-nt motif as essential for localization. These results allow insight into what constitutes an RNA localization signal and how RNA sequence elements may act in the localization process.

Elaboration of the Messenger Transport Organizer Pathway for Localization of RNA to the Vegetal Cortex ofXenopusOocytes

Previous studies demonstrated that there were two pathways, the messenger transport organizer (METRO) or early and the Vg1 or late, which function during stages 1 to 3 of oogenesis for the localization of RNAs at the vegetal cortex ofXenopusoocytes. In the present study we analyzed the properties of the METRO pathway, which localizes Xlsirt, Xcat2, and Xwnt11 RNAs to a specific region of the vegetal cortex during stage 1 of oogenesis. A combination of methodologies involving both fixed material and living oocytes was used to analyze RNA localization. We show that in early diplotene pre-stage 1 oocytes (25–50 μm in diameter) both endogenous and injected exogenous METRO RNAs translocated to multiple mitochondrial aggregates (pre-mitochondrial clouds) that surround the germinal vesicle (GV). However, by early stage 1 (diplotene oocytes, 50–200 μm), all three of the RNAs discriminated between the different clouds and translocated exclusively within the METRO of a single mitochondrial cloud. Therefore, in stage 1 diplotene oocytes there is a unique mechanism causing a change in the intrinsic property of the mitochondrial clouds which designates one of them as the RNA transport vehicle. During translocation through the cytoplasm Xlsirt and Xcat2 RNAs were detected associated with cytoplasmic particles of different morphologies. Additionally, we also found that the translocation of RNAs through the early or METRO pathway, unlike that of the late pathway, occurred in the absence of intact microtubule and actin microfilament cytoskeletal elements. This supports a cytoskeletal-independent model for localization of RNAs through the METRO pathway.

RNA Transport to the Vegetal Cortex ofXenopusOocytes

Xcat-2 RNA, a component of the germ plasm inXenopus,localizes with the mitochondrial cloud material to the vegetal cortex in stage II oocytes.Vg1 RNA also localizes to the vegetal cortex, but later in stage III/IV oocytes, using a microtubule dependent pathway. To further analyze the mechanisms involved in RNA transport,in situhybridization and autoradiography were used to follow the localization of endogenousVg1 and injectedXcat-2 transcripts in stage IV oocytes. We show thatXcat-2 is competent to localize to the vegetal cortex quite independently of the mitochondrial cloud.Xcat-2 RNA appears to use the lateVg1 localization pathway, as depolymerization of microtubules by cold or nocodazole treatment prevented translocation ofXcat-2 transcripts, but did not result in the disruption ofXcat-2 anchored in the cortex. Furthermore, RNA transport was shown to be stage dependent for bothVg1 andXcat-2 RNAs, as they did not localize in fully grown stage VI oocytes after injection. RNA sequences both required and sufficient to directXcat-2 to the vegetal cortex were mapped to a sequence of 150 nt immediately adjacent to the open reading frame and additional sequences at the end of the 3′ untranslated region. Mapping was accomplished by injecting deletion mutant transcripts into stage IV oocytes and monitoring localization by RNase protection and autoradiography. All mutants competent for translocation were also capable of cortical anchoring, suggesting that the same signal is used for both steps. We speculate that two separate RNA pathways evolved during the course ofXenopusoogenesis. One pathway, specialized for the transport of germ plasm by way of the mitochondrial cloud, occurs early to ensure the segregation of the germ cell lineage. The other, late, pathway may serve as the more general transport system for localizing RNAs involved in somatic cell differentiation.