Processes That Occur Before Second Cleavage Determine Third Cleavage Orientation in Xenopus

Experimental Cell Research. Mar, 2002  |  Pubmed ID: 11855862

As in many organisms, the first three cleavage planes of Xenopus laevis eggs form in a well-described mutually orthogonal geometry. The factors dictating this simple pattern have not been unambiguously identified. Here, we describe experiments, using static magnetic fields as a novel approach to perturb normal cleavage geometry, that provide new insight into these factors. We show that a magnetic field applied during either or both of the first two cell cycles can induce the third cell cycle mitotic apparatus (MA) at metaphase and the third cleavage plane to align nearly perpendicular to their nominal orientations without changing cell shape. These results indicate that processes occurring during the first two cell cycles primarily dictate the third cleavage plane and mitotic apparatus orientation. We discuss how mechanisms that can align the MA after it has formed are likely to be of secondary importance in determining cleavage geometry in this system.

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

Developmental Biology. Aug, 2002  |  Pubmed ID: 12142022

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.

Apparent Mitochondrial Asymmetry in Xenopus Eggs

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Apr, 2003  |  Pubmed ID: 12666203

Cell polarity is manifest along the animal/vegetal axis in eggs of the frog, Xenopus laevis. Along this axis, maternal cytoplasmic components are asymmetrically distributed and are thought to underlie specification of distinct cell fates. To ascertain the molecular identities of such cytoplasmic components, we have used a monoclonal antibody that specifically stains the vegetal hemisphere of Xenopus eggs. The antigenic protein Vp67 (vegetal protein of 67 kDa) was identified through purification and cloning as a Xenopus homolog of the mitochondrial protein dihydrolipoamide acetyltransferase, a component of the pyruvate dehydrogenase complex. The identification of Vp67 as a mitochondrial protein could indicate that populations of mitochondria are asymmetrically distributed in Xenopus eggs.

Protein Kinase A Phosphorylation Modulates Transport of the Polypyrimidine Tract-binding Protein

Proceedings of the National Academy of Sciences of the United States of America. Jul, 2003  |  Pubmed ID: 12851456

The heterogeneous nuclear ribonucleoprotein particle (hnRNP) proteins play important roles in mRNA processing in eukaryotes, but little is known about how they are regulated by cellular signaling pathways. The polypyrimidine-tract binding protein (PTB, or hnRNP I) is an important regulator of alternative pre-mRNA splicing, of viral RNA translation, and of mRNA localization. Here we show that the nucleo-cytoplasmic transport of PTB is regulated by the 3',5'-cAMP-dependent protein kinase (PKA). PKA directly phosphorylates PTB on conserved Ser-16, and PKA activation in PC12 cells induces Ser-16 phosphorylation. PTB carrying a Ser-16 to alanine mutation accumulates normally in the nucleus. However, export of this mutant protein from the nucleus is greatly reduced in heterokaryon shuttling assays. Conversely, hyperphosphorylation of PTB by coexpression with the catalytic subunit of PKA results in the accumulation of PTB in the cytoplasm. This accumulation is again specifically blocked by the S16A mutation. Similarly, in Xenopus oocytes, the phospho-Ser-16-PTB is restricted to the cytoplasm, whereas the non-Ser-16-phosphorylated PTB is nuclear. Thus, direct PKA phosphorylation of PTB at Ser-16 modulates the nucleo-cytoplasmic distribution of PTB. This phosphorylation likely plays a role in the cytoplasmic function of PTB.

Conserved and Clustered RNA Recognition Sequences Are a Critical Feature of Signals Directing RNA Localization in Xenopus Oocytes

Mechanisms of Development. Jan, 2004  |  Pubmed ID: 14706704

Although it is widely regarded that the targeting of RNA molecules to subcellular destinations depends upon the recognition of cis-elements found within their 3' untranslated regions (UTR), relatively little is known about the specific features of these cis-sequences that underlie their function. Interaction between specific repeated motifs within the 3' UTR and RNA-binding proteins has been proposed as a critical step in the localization of Vg1 RNA to the vegetal pole of Xenopus oocytes. To understand the relative contributions of repeated localization element (LE) sequences, we used comparative functional analysis of Vg1 LEs from two frog species, Xenopus laevis and Xenopus borealis. We show that clusters of repeated VM1 and E2 motifs are required for efficient localization. However, groups of either site alone are not sufficient for localization. In addition, we present evidence that the X. borealis Vg1 LE is recognized by the same set of RNA-binding proteins as the X. laevis Vg1 LE and is capable of productive interactions with the X. laevis transport machinery as it is sufficient to direct vegetal localization in X. laevis oocytes. These results suggest that clustered sets of cis-acting sites within the LE direct vegetal transport through specific interactions with the localization machinery.

Nuclear RNP Complex Assembly Initiates Cytoplasmic RNA Localization

The Journal of Cell Biology. Apr, 2004  |  Pubmed ID: 15096527

Cytoplasmic localization of mRNAs is a widespread mechanism for generating cell polarity and can provide the basis for patterning during embryonic development. A prominent example of this is localization of maternal mRNAs in Xenopus oocytes, a process requiring recognition of essential RNA sequences by protein components of the localization machinery. However, it is not yet clear how and when such protein factors associate with localized RNAs to carry out RNA transport. To trace the RNA-protein interactions that mediate RNA localization, we analyzed RNP complexes from the nucleus and cytoplasm. We find that an early step in the localization pathway is recognition of localized RNAs by specific RNA-binding proteins in the nucleus. After transport into the cytoplasm, the RNP complex is remodeled and additional transport factors are recruited. These results suggest that cytoplasmic RNA localization initiates in the nucleus and that binding of specific RNA-binding proteins in the nucleus may act to target RNAs to their appropriate destinations in the cytoplasm.

Xenopus Staufen is a Component of a Ribonucleoprotein Complex Containing Vg1 RNA and Kinesin

Development (Cambridge, England). Jul, 2004  |  Pubmed ID: 15163628

RNA localization is a key mechanism for generating cell and developmental polarity in a wide variety of organisms. We have performed studies to investigate a role for the Xenopus homolog of the double-stranded RNA-binding protein, Staufen, in RNA localization during oogenesis. We have found that Xenopus Staufen (XStau) is present in a ribonucleoprotein complex, and associates with both a kinesin motor protein and vegetally localized RNAs Vg1 and VegT. A functional role for XStau was revealed through expression of a dominant-negative version that blocks localization of Vg1 RNA in vivo. Our results suggest a central role for XStau in RNA localization in Xenopus oocytes, and provide evidence that Staufen is a conserved link between specific mRNAs and the RNA localization machinery.

Localization of RNAs to the Mitochondrial Cloud in Xenopus Oocytes Through Entrapment and Association with Endoplasmic Reticulum

Molecular Biology of the Cell. Oct, 2004  |  Pubmed ID: 15292452

The germ cell lineage in Xenopus is specified by the inheritance of germ plasm, which originates within a distinct "mitochondrial cloud" (MC) in previtellogenic oocytes. Germ plasm contains localized RNAs implicated in germ cell development, including Xcat2 and Xdazl. To understand the mechanism of the early pathway through which RNAs localize to the MC, we applied live confocal imaging and photobleaching analysis to oocytes microinjected with fluorescent Xcat2 and Xdazl RNA constructs. These RNAs dispersed evenly throughout the cytoplasm through diffusion and then became progressively immobilized and formed aggregates in the MC. Entrapment in the MC was not prevented by microtubule disruption and did not require localization to germinal granules. Immobilized RNA constructs codistributed and showed coordinated movement with densely packed endoplasmic reticulum (ER) concentrated in the MC, as revealed with Dil16(3) labeling and immunofluorescence analysis. Vg1RBP/Vera protein, which has been implicated in linking late pathway RNAs to vegetal ER, was shown to bind specifically both wild-type Xcat2 3' untranslated region and localization-defective constructs. We found endogenous Vg1RBP/Vera and Vg1RBP/Vera-green fluorescent protein to be largely excluded from the MC but subsequently to codistribute with Xcat2 and ER at the vegetal cortex. We conclude that germ line RNAs localize into the MC through a diffusion/entrapment mechanism involving Vg1RBP/Vera-independent association with ER.

Putting RNAs in the Right Place at the Right Time: RNA Localization in the Frog Oocyte

Biology of the Cell / Under the Auspices of the European Cell Biology Organization. Jan, 2005  |  Pubmed ID: 15601255

Localization of maternal mRNAs in many developing organisms provides the basis for both initial polarity during oogenesis and patterning during embryogenesis. Prominent examples of this phenomenon are found in Xenopus laevis, where localized maternal mRNAs generate developmental polarity along the animal/vegetal axis. Targeting of mRNA molecules to specific subcellular regions is a fundamental mechanism for spatial regulation of gene expression, and considerable progress has been made in defining the underlying molecular pathways.

Ribonucleoprotein Remodeling During RNA Localization

Differentiation; Research in Biological Diversity. Jul, 2007  |  Pubmed ID: 17381548

Cytoplasmic RNA localization is a means to create polarity by restricting protein expression to a discrete subcellular location. RNA localization is a multistep process that begins with the recognition of cis-acting sequences within the RNA by specific trans-factors, and RNAs are localized in ribonucleoprotein (RNP) complexes that contain both the RNA and numerous protein components. Components of the localization machinery transport the RNP complex, usually in a translationally repressed state, to a distinct subcellular region, resulting in spatially restricted gene expression. Recent efforts to identify both the cis- and trans-factors required for RNA localization have elucidated RNA-protein interactions that are remodeled during localization.

PTB/hnRNP I is Required for RNP Remodeling During RNA Localization in Xenopus Oocytes

Molecular and Cellular Biology. Jan, 2008  |  Pubmed ID: 18039852

Transport of specific mRNAs to defined regions within the cell cytoplasm is a fundamental mechanism for regulating cell and developmental polarity. In the Xenopus oocyte, Vg1 RNA is transported to the vegetal cytoplasm, where localized expression of the encoded protein is critical for embryonic polarity. The Vg1 localization pathway is directed by interactions between key motifs within Vg1 RNA and protein factors recognizing those RNA sequences. We have investigated how RNA-protein interactions could be modulated to trigger distinct steps in the localization pathway and found that the Vg1 RNP is remodeled during cytoplasmic RNA transport. Our results implicate two RNA-binding proteins with key roles in Vg1 RNA localization, PTB/hnRNP I and Vg1RBP/vera, in this process. We show that PTB/hnRNP I is required for remodeling of the interaction between Vg1 RNA and Vg1RBP/vera. Critically, mutations that block this remodeling event also eliminate vegetal localization of the RNA, suggesting that RNP remodeling is required for localization.

Multiple Kinesin Motors Coordinate Cytoplasmic RNA Transport on a Subpopulation of Microtubules in Xenopus Oocytes

Developmental Cell. Sep, 2008  |  Pubmed ID: 18771961

RNA localization is a widely conserved mechanism for generating cellular asymmetry. In Xenopus oocytes, microtubule-dependent transport of RNAs to the vegetal cortex underlies germ layer patterning. Although kinesin motors have been implicated in this process, the apparent polarity of the microtubule cytoskeleton has pointed instead to roles for minus-end-directed motors. To resolve this issue, we have analyzed participation of kinesin motors in vegetal RNA transport and identified a direct role for Xenopus kinesin-1. Moreover, in vivo interference and biochemical experiments reveal a key function for multiple motors, specifically kinesin-1 and kinesin-2, and suggest that these motors may interact during transport. Critically, we have discovered a subpopulation of microtubules with plus ends at the vegetal cortex, supporting roles for these kinesin motors in vegetal RNA transport. These results provide a new mechanistic basis for understanding directed RNA transport within the cytoplasm.

VISIONS: the Art of Science

Molecular Reproduction and Development. Dec, 2009  |  Pubmed ID: 19834979

Preparation of a Highly Active Cell-free Translation System from Immature Xenopus Laevis Oocytes

Methods (San Diego, Calif.). May, 2010  |  Pubmed ID: 20123127

Understanding mechanisms of post-transcriptional control of gene expression has come under much scrutiny in recent years. A key question in this field is how the translation of specific mRNAs is activated or repressed both spatially and temporally in a given cell. In oocytes of the frog Xenopus laevis a number of mRNAs are localized early in oogenesis and subsequently translated at later stages. We have developed a highly active cell-free translation system from oocytes in the early stages of oogenesis that is applicable to the study of translation and translational control of both endogenous and exogenous mRNAs.

Visualization of MRNA Localization in Xenopus Oocytes

Methods in Molecular Biology (Clifton, N.J.). 2011  |  Pubmed ID: 21431735

Visualization of in vivo mRNA localization provides a tool for understanding steps in the mechanism of transport. Here we detail a method of fluorescently labeling mRNA transcripts and microinjecting them into Xenopus laevis oocytes followed with imaging by confocal microscopy. This technique overcomes a significant hurdle of imaging RNA in the frog oocyte while providing a rapid method of visualizing mRNA localization in high resolution.

Molecular Motors: Directing Traffic During RNA Localization

Critical Reviews in Biochemistry and Molecular Biology. Jun, 2011  |  Pubmed ID: 21476929

RNA localization, the enrichment of RNA in a specific subcellular region, is a mechanism for the establishment and maintenance of cellular polarity in a variety of systems. Ultimately, this results in a universal method for spatially restricting gene expression. Although the consequences of RNA localization are well-appreciated, many of the mechanisms that are responsible for carrying out polarized transport remain elusive. Several recent studies have illuminated the roles that molecular motor proteins play in the process of RNA localization. These studies have revealed complex mechanisms in which the coordinated action of one or more motor proteins can act at different points in the localization process to direct RNAs to their final destination. In this review, we discuss recent findings from several different systems in an effort to clarify pathways and mechanisms that control the directed movement of RNA.