Xenopus Oocyte Nuclei Research Articles

Long-term association of a transcription factor with its chromatin binding site can stabilize gene expression and cell fate commitment

Some lineage-determining transcription factors are overwhelmingly important in directing embryonic cells to a particular differentiation pathway, such as Ascl1 for nerve. They also have an exceptionally strong ability to force cells to change from an unrelated pathway to one preferred by their action. Transcription factors are believed to have a very short residence time of only a few seconds on their specific DNA or chromatin-binding sites. We have developed a procedure in which DNA containing one copy of the binding site for the neural-inducing factor Ascl1 is injected directly into a Xenopus oocyte nucleus which has been preloaded with a limiting amount of the Ascl1 transcription factor protein. This is followed by a further injection of DNA as a competitor, either in a plasmid or in chromosomal DNA, containing the same binding site but with a different reporter. Importantly, expression of the reporter provides a measure of the function of the transcription factor in addition to its residence time. The same long residence time and resistance to competition are seen with the estrogen receptor and its DNA response elements. We find that in this nondividing oocyte, the nerve-inducing factor Ascl1 can remain bound to a specific chromatin site for hours or days and thereby help to stabilize gene expression. This stability of transcription factor binding to chromatin is a necessary part of its action because removal of this factor causes discontinuation of its effect on gene expression. Stable transcription factor binding may be a characteristic of nondividing cells.

Nanoscale electrostatic gating of molecular transport through nuclear pore complexes as probed by scanning electrochemical microscopy.

The nuclear pore complex (NPC) is a large protein nanopore that solely mediates molecular transport between the nucleus and cytoplasm of a eukaryotic cell. There is a long-standing consensus that selective transport barriers of the NPC are exclusively based on hydrophobic repeats of phenylalanine and glycine (FG) of nucleoporins. Herein, we reveal experimentally that charged residues of amino acids intermingled between FG repeats can modulate molecular transport through the NPC electrostatically and in a pathway-dependent manner. Specifically, we investigate the NPC of the Xenopus oocyte nucleus to find that excess positive charges of FG-rich nucleoporins slow down passive transport of a polycationic peptide, protamine, without affecting that of a polyanionic pentasaccharide, Arixtra, and small monovalent ions. Protamine transport is slower with a lower concentration of electrolytes in the transport media, where the Debye length becomes comparable to the size of water-filled spaces among the gel-like network of FG repeats. Slow protamine transport is not affected by the binding of a lectin, wheat germ agglutinin, to the peripheral route of the NPC, which is already blocked electrostatically by adjacent nucleoporins that have more cationic residues than anionic residues and even FG dipeptides. The permeability of NPCs to the probe ions is measured by scanning electrochemical microscopy using ion-selective tips based on liquid/liquid microinterfaces and is analysed by effective medium theory to determine the sizes of peripheral and central routes with distinct protamine permeability. Significantly, nanoscale electrostatic gating at the NPC can be relevant not only chemically and biologically, but also biomedically for efficient nuclear import of genetically therapeutic substances.

Isolation and Analysis of Xenopus Germinal Vesicles.

The giant nucleus or germinal vesicle (GV) of Xenopus oocytes provides an unusual opportunity to analyze nuclear structure and function in exquisite detail by light microscopy. Detailed here are two rapid procedures for using manually isolated GVs in combination with fluorescent reporter proteins to investigate the lampbrush chromosomes and nuclear bodies of oocytes. One procedure provides spreads of nuclear components in an unfixed and life-like, although not living, form. The other describes the isolation of intact, functional GVs directly into mineral oil offering possibilities for direct observation of nuclear dynamics.

Studying nuclear functions of aminoacyl tRNA synthetases

Aminoacyl tRNA synthetases (AARSs) are best known for their essential role in translation in the cytoplasm. The concept that AARSs also exist in the nucleus started to draw attention around the turn of the new millennium, when aminoacylated tRNAs were first found in the nuclei of Xenopus oocytes. It is now expected that all cytoplasmic AARSs are present in the nucleus. In addition to tRNA aminoacylation, nuclear AARSs were found to regulate a spectrum of biological processes and responses, with many AARSs functioning through regulation at the level of gene transcription. In this paper, we focus on describing methods that have been successfully implemented to study AARSs in transcriptional regulation. These include a cell fractionation assay to detect nuclear localization, an in vitro DNA-cellulose pull-down assay to determine DNA binding capacity, and a chromatin immunoprecipitation (ChIP)-DNA deep sequencing assay to identify DNA binding sites. Application of these methods would expand our understanding of AARS functions and reveal critical insights on the coordination of gene transcription and translation.

A deep proteomics perspective on CRM1-mediated nuclear export and nucleocytoplasmic partitioning.

CRM1 is a highly conserved, RanGTPase-driven exportin that carries proteins and RNPs from the nucleus to the cytoplasm. We now explored the cargo-spectrum of CRM1 in depth and identified surprisingly large numbers, namely >700 export substrates from the yeast S. cerevisiae, ≈1000 from Xenopus oocytes and >1050 from human cells. In addition, we quantified the partitioning of ≈5000 unique proteins between nucleus and cytoplasm of Xenopus oocytes. The data suggest new CRM1 functions in spatial control of vesicle coat-assembly, centrosomes, autophagy, peroxisome biogenesis, cytoskeleton, ribosome maturation, translation, mRNA degradation, and more generally in precluding a potentially detrimental action of cytoplasmic pathways within the nuclear interior. There are also numerous new instances where CRM1 appears to act in regulatory circuits. Altogether, our dataset allows unprecedented insights into the nucleocytoplasmic organisation of eukaryotic cells, into the contributions of an exceedingly promiscuous exportin and it provides a new basis for NES prediction.

Finding the unexpected--how we identified a second class of introns and the U12-dependent spliceosome.

Finding the unexpected--how we identified a second class of introns and the U12-dependent spliceosome.

Selective nuclear export of specific classes of mRNA from mammalian nuclei is promoted by GANP.

The nuclear phase of the gene expression pathway culminates in the export of mature messenger RNAs (mRNAs) to the cytoplasm through nuclear pore complexes. GANP (germinal- centre associated nuclear protein) promotes the transfer of mRNAs bound to the transport factor NXF1 to nuclear pore complexes. Here, we demonstrate that GANP, subunit of the TRanscription-EXport-2 (TREX-2) mRNA export complex, promotes selective nuclear export of a specific subset of mRNAs whose transport depends on NXF1. Genome-wide gene expression profiling showed that half of the transcripts whose nuclear export was impaired following NXF1 depletion also showed reduced export when GANP was depleted. GANP-dependent transcripts were highly expressed, yet short-lived, and were highly enriched in those encoding central components of the gene expression machinery such as RNA synthesis and processing factors. After injection into Xenopus oocyte nuclei, representative GANP-dependent transcripts showed faster nuclear export kinetics than representative transcripts that were not influenced by GANP depletion. We propose that GANP promotes the nuclear export of specific classes of mRNAs that may facilitate rapid changes in gene expression.

The micro and macro of RNA function

The central dogma uniquely positions RNA as the key molecular player for translating genetic software into protein hardware. But it has become increasingly clear that RNA also plays an essential regulatory role within cells. MicroRNAs in particular have emerged as ubiquitous and still poorly understood molecules involved in the control of a wide variety of biological processes. RNA has also recently been shown to have important roles in structural assembly within cells. A diverse set of speakers at the Minisymposium on Micro- and Coding RNA highlighted some of these new and often unexpected roles of RNA across a range of length scales. Tracy Johnson (University of California–San Diego) discussed her lab's work on cotranscriptional mRNA splicing. Her talk underscored the importance of ATP-dependent RNA helicases in rearranging RNA secondary structure and the integral role for chromatin modification in coordinating RNA rearrangements with transcription in space and time. Albert Rosana (Owttrim lab, University of Alberta) also discussed RNA helicases within the fascinating context of temperature fluctuations of cyanobacteria. He described his work on the altered expression of the oligomeric RNA helicase CrhR as a temperature stress response. His work provides new insights into RNA regulation, suggesting CrhR autoregulates its expression through a combination of RNA processing and RNA–protein stabilization mechanisms, which in turn leads to temperature-dependent regulation of small regulatory RNAs. Several presentations explored how RNAs play a role in the assembly of large-scale intracellular structures. Magdalena Strzelecka (Heald lab, University of California–Berkeley) discussed her work on RNAs in the assembly of the mitotic spindle. She utilized Xenopus egg extracts and next-generation sequencing approaches to demonstrate a surprising role for splicing factors in mitosis. This suggests the intriguing possibility that RNA processing plays a regulatory role in cell division. Moving to even larger scales, Cliff Brangwynne (Princeton University) discussed his lab's work on the biophysics of ribonucleoprotein (RNP) bodies—large, non–membrane bound assemblies that behave as liquid-phase droplets of RNA and protein. This work exploits the nucleus of Xenopus oocytes to study the nucleolus, an RNP droplet involved in ribosome biogenesis. The presentation revealed some unexpected biophysical consequences of the large length scales involved, with important implications for nuclear RNP organization and function. The nucleolus was also the central theme of a talk by Thoru Pederson (University of Massachusetts Medical School), who summarized his lab's recently published work on a subset of microRNAs localized in the nucleolus of rat myoblasts. He also reported new results on the nucleolar presence of certain mRNAs in these cells, proposing there might be microRNA–mRNA interactions in the nucleolus to pre-set the translational status of exported mRNAs. The remaining talk of the session, by Rebecca Holmes (Tollervey lab, University of Edinburgh), focused on the yeast protein Npl3, a member of the large SR family of RNA-binding proteins with diverse functions in pre-mRNA processing. She described experiments utilizing in vivo cross-linking and deep sequencing, which revealed the binding of Npl3 to a surprising variety of RNA species. Her data suggest an unexpected role for Npl3 in regulating noncoding RNAs (ncRNAs), with consequences for the expression of surrounding genes. These results raise the possibility that other previously described functions of Npl3 may, in fact, be consequences of Npl3 regulation of ncRNAs.

Nuclear actin and transcriptional activation

Differentiated cells do not revert to an embryonic state in normal development. However, the method called nuclear reprogramming enables these differentiated cells to be reversed to an embryonic state. One essential event in the reprogramming process is reactivation of embryonic genes such as Oct4 (also known as Pou5f1). This reprogramming of transcriptional programs can be achieved by transplantation of mammalian somatic nuclei to the giant Xenopus laevis oocyte nucleus, referred to as the germinal vesicle (GV). Factors and mechanisms responsible for this transcriptional reprogramming have not been elucidated. Recently, we have found that a polymerized form of actin is abundantly present in nuclei transplanted into the Xenopus oocyte nucleus and plays an important role in transcriptional reactivation of Oct4. This study emphasizes a significant contribution of nuclear actin in transcriptional activation. Here, we discuss possible roles of nuclear actin in Xenopus oocytes and in other cell types in the context of transcriptional activation.

Nuclear actin and transcriptional activation.

Differentiated cells do not revert to an embryonic state in normal development. However, the method called nuclear reprogramming enables these differentiated cells to be reversed to an embryonic state. One essential event in the reprogramming process is reactivation of embryonic genes such as Oct4 (also known as Pou5f1). This reprogramming of transcriptional programs can be achieved by transplantation of mammalian somatic nuclei to the giant Xenopus laevis oocyte nucleus, referred to as the germinal vesicle (GV). Factors and mechanisms responsible for this transcriptional reprogramming have not been elucidated. Recently, we have found that a polymerized form of actin is abundantly present in nuclei transplanted into the Xenopus oocyte nucleus and plays an important role in transcriptional reactivation of Oct4. This study emphasizes a significant contribution of nuclear actin in transcriptional activation. Here, we discuss possible roles of nuclear actin in Xenopus oocytes and in other cell types in the context of transcriptional activation.

The nuclear experience of CPEB: Implications for RNA processing and translational control

CPEB is a sequence-specific RNA binding protein that promotes polyadenylation-induced translation in early development, during cell cycle progression and cellular senescence, and following neuronal synapse stimulation. It controls polyadenylation and translation through other interacting molecules, most notably the poly(A) polymerase Gld2, the deadenylating enzyme PARN, and the eIF4E-binding protein Maskin. Here, we report that CPEB shuttles between the nucleus and cytoplasm and that its export occurs via the CRM1-dependent pathway. In the nucleus of Xenopus oocytes, CPEB associates with lampbrush chromosomes and several proteins involved in nuclear RNA processing. CPEB also interacts with Maskin in the nucleus as well as with CPE-containing mRNAs. Although the CPE does not regulate mRNA export, it influences the degree to which mRNAs are translationally repressed in the cytoplasm. Moreover, CPEB directly or indirectly mediates the alternative splicing of at least one pre-mRNA in mouse embryo fibroblasts as well as certain mouse tissues. We propose that CPEB, together with Maskin, binds mRNA in the nucleus to ensure tight translational repression upon export to the cytoplasm. In addition, we propose that nuclear CPEB regulates specific pre-mRNA alternative splicing.

Small Cajal body-specific RNAs of Drosophila function in the absence of Cajal bodies.

During their biogenesis small nuclear RNAs (snRNAs) undergo multiple covalent modifications that require guide RNAs to direct methylase and pseudouridylase enzymes to the appropriate nucleotides. Because of their localization in the nuclear Cajal body (CB), these guide RNAs are known as small CB-specific RNAs (scaRNAs). Using a fluorescent primer extension technique, we mapped the modified nucleotides in Drosophila U1, U2, U4, and U5 snRNAs. By fluorescent in situ hybridization (FISH) we showed that seven Drosophila scaRNAs are concentrated in easily detectable CBs. We used two assays based on Xenopus oocyte nuclei to demonstrate that three of these Drosophila scaRNAs do, in fact, function as guide RNAs. In flies null for the CB marker protein coilin, CBs are absent and there are no localized FISH signals for the scaRNAs. Nevertheless, biochemical experiments show that scaRNAs are present at normal levels and snRNAs are properly modified. Our experiments demonstrate that several scaRNAs are concentrated as expected in the CBs of wild-type Drosophila, but they function equally well in the nucleoplasm of mutant flies that lack CBs. We propose that the snRNA modification machinery is not limited to CBs, but is dispersed throughout the nucleoplasm of cells in general.

Nuclear myosin 1 is in complex with mature rRNA transcripts and associates with the nuclear pore basket

In rRNA biogenesis, nuclear myosin 1 (NM1) and actin synergize to activate rRNA gene transcription. Evidence that actin is in preribosomal subunits and NM1 may control rRNA biogenesis post-transcriptionally prompted us to investigate whether NM1 associates with and accompanies rRNA to nuclear pores (NPC). Ultracentrifugation on HeLa nucleolar extracts showed RNA-dependent NM1 coelution with preribosomal subunits. In RNA immunoprecipitations (RIPs), NM1 coprecipitated with pre-rRNAs and 18S, 5.8S, and 28S rRNAs, but failed to precipitate 5S rRNA and 7SL RNA. In isolated nuclei and living HeLa cells, NM1 or actin inhibition and selective alterations in actin polymerization impaired 36S pre-rRNA processing. Immunoelectron microscopy (IEM) on sections of manually isolated Xenopus oocyte nuclei showed NM1 localization at the NPC basket. Field emission scanning IEM on isolated nuclear envelopes and intranuclear content confirmed basket localization and showed that NM1 decorates actin-rich pore-linked filaments. Finally, RIP and successive RIPs (reRIPs) on cross-linked HeLa cells demonstrated that NM1, CRM1, and Nup153 precipitate same 18S and 28S rRNAs but not 5S rRNA. We conclude that NM1 facilitates maturation and accompanies export-competent preribosomal subunits to the NPC, thus modulating export.

Cargo surface hydrophobicity is sufficient to overcome the nuclear pore complex selectivity barrier

To fulfil their function, nuclear pore complexes (NPCs) must discriminate between inert proteins and nuclear transport receptors (NTRs), admitting only the latter. This specific permeation is thought to depend on interactions between hydrophobic patches on NTRs and phenylalanine-glycine (FG) or related repeats that line the NPC. Here, we tested this premise directly by conjugating different hydrophobic amino-acid analogues to the surface of an inert protein and examining its ability to cross NPCs unassisted by NTRs. Conjugation of as few as four hydrophobic moieties was sufficient to enable passage of the protein through NPCs. Transport of the modified protein proceeded with rates comparable to those measured for the innate protein when bound to an NTR and was relatively insensitive both to the nature and density of the amino acids used to confer hydrophobicity. The latter observation suggests a non-specific, small, and plant interaction network between cargo and FG repeats.

Nuclear and cytoplasmic organization in Xenopus oocytes after disruption of actin filaments by latrunculin

Actin-containing filaments have been visualized inside Xenopus oocyte nuclei by a combination of fluorescence and transmission electron microscopy. It was shown that these filaments contact nucleoli, spherical bodies, and nuclear pore complexes. The incubation of oocytes with actin-depolymerizing agent, latrunculin, caused membrane vesiculation in cytoplasm and the disruption of nucleoplasm and the integrity of the nuclear envelope. We suggest that actin-containing filaments are important cell components involved in the regulation of nucleus-cytoplasm interactions, as well as of cellular transport of components during the growth of Xenopus oocytes.

Reticulon 4a/NogoA locates to regions of high membrane curvature and may have a role in nuclear envelope growth

Reticulon 4a (Rtn4a) is a membrane protein that shapes tubules of the endoplasmic reticulum (ER). The ER is attached to the nuclear envelope (NE) during interphase and has a role in post mitotic/meiotic NE reassembly. We speculated that Rtn4a has a role in NE dynamics. Using immuno-electron microscopy we found that Rtn4a is located at junctions between membranes in the cytoplasm, and between cytoplasmic membranes and the outer nuclear membrane in growing Xenopus oocyte nuclei. We found that during NE assembly in Xenopus egg extracts, Rtn4a localises to the edges of membranes that are flattening onto the chromatin. These results demonstrate that Rtn4a locates to regions of high membrane curvature in the ER and the assembling NE. Previously it was shown that incubation of egg extracts with antibodies against Rtn4a caused ER to form into large vesicles instead of tubules. To test whether Rtn4a contributes to NE assembly, we added the same Rtn4a antibody to nuclear assembly reactions. Chromatin was enclosed by membranes containing nuclear pore complexes, but nuclei did not grow. Instead large sacs of ER membranes attached to, but did not integrate into the NE. It is possible therefore that Rtn4a may have a role in NE assembly.

Factors associated with a purine-rich exonic splicing enhancer sequence in Xenopus oocyte nucleus

Purine-rich exonic splicing enhancers (ESEs) stimulate splicing of the adjacent introns with suboptimal splice sites. To elucidate the mechanism regarding ESEs, factors specifically associated with ESEs in HeLa cell nuclear extracts were previously investigated, and shown to include SR (serine/arginine-rich) proteins. However, factors associated with ESEs in vivo have not yet been explored. Here we show that a GAA repeat RNA sequence, a typical ESE, is associated in Xenopus oocyte nuclei with at least one SR protein, SF2/ASF, as was expected. Moreover, components of SF3a/b complexes, U2 snRNA, and U2AF65 were also found to be associated with the ESE in the nucleus. Since SF3a/b complexes are the constituents of the 17S U2 snRNP, these results suggest that the 17S U2 snRNP is associated with the ESE in the nucleus, probably through bridging interactions of U2AF and SR proteins. The identified factors may represent a functional splicing enhancer complex in vivo.

Pore-linked filaments in anura spermatocyte nuclei

Pore-linked filaments were visualized in spreads of anuran spermatocyte nuclei using transmission electron microscope. We used Odontophrynus diplo and tetraploid species having the tetraploid frogs reduced metabolic activities. The filaments with 20-40 nm width are connected to a ring component of the nuclear pore complex with 90-120 nm and extend up to 1 microm (or more) into the nucleus. The filaments are curved and connect single or neighboring pores. The intranuclear filaments are associated with chromatin fibers and related to RNP particles of 20-25 nm and spheroidal structures of 0.5 microm, with variations. The aggregates of several neighboring pores with the filaments are more commonly observed in 4n nuclei. We concluded that the intranuclear filaments may correspond to the fibrillar network described in Xenopus oocyte nucleus being probably related to RNA transport. The molecular basis of this RNA remains elusive. Nevertheless, the morphological aspects of the spheroidal structures indicate they could correspond to nucleolar chromatin or to nucleolus-derived structures. We also speculate whether the complex aggregates of neighboring pores with intranuclear filaments may correspond to pore clustering previously described in these tetraploid animals using freeze-etching experiments.

Changes in Nucleoporin Domain Topology in Response to Chemical Effectors

Nucleoporins represent the molecular building blocks of nuclear pore complexes (NPCs), which mediate facilitated macromolecular trafficking between the cytoplasm and nucleus of eukaryotic cells. Phenylalanine-glycine (FG) repeat motifs are found in about one-third of the nucleoporins, and they provide major binding or docking sites for soluble transport receptors. We have shown recently that localization of the FG-repeat domains of vertebrate nucleoporins Nup153 and Nup214 within the NPC is influenced by its transport state. To test whether chemical effectors, such as calcium and ATP, influence the localization of the FG-repeat domains of Nup153 and Nup214 within the NPC, we performed immuno-electron microscopy of Xenopus oocyte nuclei using domain-specific antibodies against Nup153 and Nup214, respectively. Ca 2+ and ATP are known to induce conformational changes in the NPC architecture, especially at the cytoplasmic face, but also at the nuclear basket of the NPC. We have found concentrations of calcium in the micromolar range or 1 mM ATP in the surrounding buffer leaves the spatial distribution of the FG-repeat of Nup153 and Nup214 largely unchanged. In contrast, ATP depletion, calcium store depletion by EGTA or thapsigargin, and high concentrations of divalent cation (i.e. 2 mM Ca 2+ and 2 mM Mg 2+) constrain the distribution of the FG-repeats of Nup153 and Nup214. Our data suggest that the location of the FG-repeat domains of Nup153 and Nup214 is sensitive to chemical changes within the near-field environment of the NPC.

A selective block of nuclear actin export stabilizes the giant nuclei of Xenopus oocytes

Actin is a major cytoskeletal element and is normally kept cytoplasmic by exportin 6 (Exp6)-driven nuclear export. Here, we show that Exp6 recognizes actin features that are conserved from yeast to human. Surprisingly however, microinjected actin was not exported from Xenopus laevis oocyte nuclei, unless Exp6 was co-injected, indicating that the pathway is inactive in this cell type. Indeed, Exp6 is undetectable in oocytes, but is synthesized from meiotic maturation onwards, which explains how actin export resumes later in embryogenesis. Exp6 thus represents the first example of a strictly developmentally regulated nuclear transport pathway. We asked why Xenopus oocytes lack Exp6 and observed that ectopic application of Exp6 renders the giant oocyte nuclei extremely fragile. This effect correlates with the selective disappearance of a sponge-like intranuclear scaffold of F-actin. These nuclei have a normal G2-phase DNA content in a volume 100,000 times larger than nuclei of somatic cells. Apparently, their mechanical integrity cannot be maintained by chromatin and the associated nuclear matrix, but instead requires an intranuclear actin-scaffold.