Oocyte RNA Research Articles

Injection of exogenous RNA in amphibian oocytes leads to RNA level fluctuations which are sensitive to cordycepin, an RNA chain elongation terminator

Using an in vivo heterologous system to study the stability of Xenopus laevis RNA injected into axolotl ( Ambystoma mexicanum) fertilized eggs, we have previously observed unexpected fluctuations in RNA level during early development [Andéol et al., Differentiation 63 (1998) 69–79]. In this study, we further characterize this phenomenon and establish its existence during axolotl and Xenopus oogenesis, suggesting a phylogenetically conserved mechanism. The phenomenon can occur with a variety of exogenous sense and antisense substrates. RNase protection experiments establish that most of the molecules have the same polarity as the initially injected RNA. In addition, trace amount of complementary RNA (cRNA) can be detected the injected samples. Cordycepin prevent increases in RNA levels indicating the involvement of an RNA synthesis. These results indicate the existence of an in vivo post-transcriptional RNA amplification mechanism during the early development of amphibians. To cite this article: N. Montreau et al., C. R. Biologies 326 (2003).

Localization of RNAs in oocytes of Eleutherodactylus coqui, a direct developing frog, differs from Xenopus laevis

Eleutherodactylus coqui develops directly on land to a frog. The large 3.5-mm oocyte of E. coqui has enough yolk to allow development without a feeding tadpole. In the smaller Xenopus laevis oocyte, 1.3 mm in diameter, mRNAs involved in germ layer formation, such as VegT and Vg1, are localized to the vegetal cortex of the oocyte. We hypothesized that an animal shift has occurred in the localization of the E. coqui Orthologs of VegT and Vg1 due to the large egg size. Through a combination of degenerate reverse transcriptase polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE), we cloned 1634 bp of EcVegT and 1377 bp of EcVg1. Northern blot analysis shows that the lengths of these transcripts are 2.5 kb and 1.3 kb, respectively. This result suggests that we have obtained the complete Vg1 transcript, although this transcript has an extremely short 3' untranslated region compared with X. laevis, 256 bp and 1268 bp, respectively. Zygotic expression of EcVegT closely resembles that of VegT, supporting their orthology. Radioactive RT-PCR and in situ hybridization demonstrated the presence of EcVegT and EcVg1 predominantly near the animal pole of the oocyte. RT-PCR showed that the animal blastomeres, formed from the first horizontal cleavage, inherit half of the EcVegT and EcVg1 transcripts, although they contain only about 1% of the embryo volume. Our results indicate major differences between the molecular organization of the eggs of X. laevis and E. coqui.

Embryonic expression of Xenopus laevis SOX7

SOX7, first described in Xenopus laevis by Shiozawa et al. [Biochim. Biophys. Acta 1309(1996)73], is a member, along with SOXs 17 and 18, of the F subgroup of SOX-type transcription factors. As part of a study of maternal SOX proteins that may modulate β-catenin signaling, we isolated a XSOX7 cDNA from oocyte RNA and examined the pattern of XSOX7 expression during early development. While present maternally cell-type specific expression was first observed in the ciliated cells within the epidermis of early neurula stage embryos. As development proceeds, the pattern of XSOX7 expression becomes increasingly complex. XSOX7 is expressed in the aortic arch, the olfactory pit, the stomodeal depression, the procardiac tube, within cells of the developing embryonic vasculature, in the notochord, and within the hindbrain. XSOX7 expression continues within the hindbrain in 3-day old (∼stage 40) larvae. Given its widespread expression, XSOX7 is likely to be involved in a number of developmental processes.

Nuclear RanGTP is not required for targeting small nucleolar RNAs to the nucleolus.

The small GTPase Ran is the central regulator of macromolecular transport between the cytoplasm and the nucleus. Recent work has suggested that RanGTP also plays an important role in regulating some intra-nuclear processes. In this study, we have investigated whether RanGTP is required for the intra-nuclear transport of RNAs. Specifically, we directly analyzed the nucleolar localization of Box C/D and Box H/ACA small nucleolar RNAs (snoRNAs) in mammalian (tsBN2) cells, Saccharomyces cerevisiae and Xenopus oocytes under conditions that deplete nuclear RanGTP and prevent RNA export to the cytoplasm. Our data suggest that depletion of nuclear RanGTP does not significantly alter the nucleolar localization of U3 snoRNA in tsBN2 cells. Complementary studies in the budding yeast S. cerevisiae using conditional Ran mutants as well as mutants in Ran regulatory proteins also indicate that disruption of the Ran gradient or of Ran itself does not detectably affect the nucleolar localization of snoRNAs. Finally, microinjection into Xenopus oocytes was used to clearly demonstrate that a specific pool of snoRNAs could still be efficiently targeted to the nucleolus even when the RanGTP gradient was disrupted by microinjection of mutant Ran proteins. Taken together, our data from three phylogenetically distinct experimental systems suggest that nuclear RanGTP, which is essential for trafficking of RNAs between the nuclear and cytoplasmic compartments, is not required for nuclear retention or nucleolar localization of snoRNAs.

UUCAC- and Vera-Dependent Localization of VegT RNA in Xenopus Oocytes

Localized mRNAs are directed to their destinations by “localization elements” (LEs) in their 3′UTRs. LEs harbor multiple, functionally redundant localization “signals.” These signals are poorly defined, hence it is unclear whether the signals—and their cognate factors—are unique to each RNA or employed generally. Five “E2s” (UUCACs) in the 366 nt Vg1 LE (VgLE) direct this transcript to the vegetal pole of Xenopus oocytes via the binding of a protein—Vera/Vg1RBP/ZBP [1–3]. Here we show that a different vegetal RNA, VegT, employs the same signal and factor. Five E2s within a 440 nt subregion (VegT440) of the VegT 3′UTR predict its LE and are both necessary and sufficient (in the context of antisense VegT440) for directing localization. The E2s in VegT440 and VgLE function similarly to recruit Vera protein: (1) in both contexts, E2 nt substitutions partially (UU to AC) or completely (CA to UG) inhibit localization in accordance with the sequence selectivity of Vera protein for E2s; (2) VegT440 and VgLE crosscompete, in an E2-dependent manner, for localization and Vera binding; (3) injection of anti-Vera antibody into oocytes inhibits localization of both injected transcripts. These findings imply that general localization signals traffic diverse RNAs.

Hypophosphorylation of Poly(A) Polymerase and Increased Polyadenylation Activity Are Associated with Human Immunodeficiency Virus Type 1 Vpr Expression

The HIV-1 encoded accessory protein, viral protein R (Vpr) is responsible for several biological effects in HIV-1-infected cells including nuclear transport of the preintegration complex, activation of long terminal repeat (LTR)-mediated transcription, and the induction of cell-cycle arrest and apoptosis. Vpr's ability to arrest cells at the G2 phase of the cell cycle is due to the inactivation of p34cdc2 cyclin B complex, resulting in hypophosphorylation of substrates involved in cell-cycle progression from G2 to mitosis (M). Poly(A) polymerase (PAP), the enzyme responsible for poly(A) addition to primary transcripts, contains multiple consensus phosphorylation sites for p34cdc2 cyclin B kinase that regulates its catalytic activity. We investigated the effects of Vpr on the activity of PAP in Jurkat cells using a superinfection system. Superinfection of cells using Vpr+ vesicular stomatitis virus G protein (VSV-G)-pseudotyped virus caused a complete dephosphorylation of PAP. Cotransfection studies in 293T cells and Xenopus oocyte RNA injection experiments mirrored these effects. Vpr's dramatic effect on PAP dephosphorylation was reflected in enhanced polyadenylation activity in PAP activity assays. HIV-1 Vpr appears to enhance processes that are coupled to transcription such as polyadenylation and could ultimately prove to optimize HIV-1 replication and contribute to HIV-1 pathogenesis.

Functional and molecular characterization of the K-Cl cotransporter of Xenopus laevis oocytes.

The K-Cl cotransporters (KCCs) have a broad range of physiological roles, in a number of cells and species. We report here that Xenopus laevis oocytes express a K-Cl cotransporter with significant functional and molecular similarity to mammalian KCCs. Under isotonic conditions, defolliculated oocytes exhibit a Cl(-)-dependent (86)Rb(+) uptake mechanism after activation by the cysteine-reactive compounds N-ethylmaleimide (NEM) and mercuric chloride (HgCl(2)). The activation of this K-Cl cotransporter by cell swelling is prevented by inhibition of protein phosphatase-1 with calyculin A; NEM activation of the transporter was not blocked by phosphatase inhibition. Kinetic characterization reveals apparent values for the Michaelis-Menten constant of 27.7 +/- 3.0 and 15.4 +/- 4.7 mM for Rb(+) and Cl(-), respectively, with an anion selectivity for K(+) transport of Cl(-) = PO(4)(3-) = Br(-) > I(-) > SCN(-) > gluconate. The oocyte K-Cl cotransporter was sensitive to several inhibitors, including loop diuretics, with apparent half-maximal inhibition values of 200 and 500 microM for furosemide and bumetanide, respectively. A partial cDNA encoding the Xenopus K-Cl cotransporter was cloned from oocyte RNA; the corresponding transcript is widely expressed in Xenopus tissues. The predicted COOH-terminal protein fragment exhibited particular homology to the KCC1/KCC3 subgroup of the mammalian KCCs, and the functional characteristics are the most similar to those of KCC1 (Mercado A, Song L, Vazquez N, Mount DB, and Gamba G. J Biol Chem 275: 30326--30334, 2000).

Splicing Factors SRp20 and 9G8 Promote the Nucleocytoplasmic Export of mRNA

We have uncovered a novel function for two members of the SR protein family in mRNA export. Using UV cross-linking, transient transfection, and Xenopus oocyte microinjection, we find that the nucleocytoplasmic shuttling proteins SRp20 and 9G8 interact specifically with a 22-nt RNA element from the histone H2a gene to promote the export of intronless RNAs in both mammalian cells and Xenopus oocytes. Antibodies to SRp20 or 9G8 eliminate RNA binding and significantly inhibit the export of RNAs carrying the element from oocyte nuclei. Our observation that SRp20 and 9G8 can be UV cross-linked to polyadenylated RNA in both the nucleus and cytoplasm of HeLa cells suggests a more general role for these SR proteins in mRNA export.

RNA localization in Xenopus oocytes.

Because of the relatively large size of Xenopus oocytes (up to 1.4 mm in diameter by the end of stage VI), their ease of injection and manipulation, and their clearly defined animal-vegetal polarity, this system represents one of the most intensively studied systems for RNA localization. One of the first screens for localized RNAs was performed on Xenopus eggs and oocytes, and identified both animally and vegetally localized RNAs (Rebagliati et al. 1985). Since then, several different techniques have identified a number of additional RNAs, whose functions are quite varied (see King et al. 1999; Mowry and Cote 1999). In addition, trans-acting factors have been described that appear to play important roles in localizing RNAs in oocytes. The role cytoskeletal elements play in localizing various RNAs has also been examined. In short, the Xenopus oocyte system can be considered in many ways a model system for understanding how RNAs are intracellularly localized.KeywordsXenopus OocyteGerminal VesicleGerm PlasmXenopus Laevis OocyteLate PathwayThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

RNA localization and germ cell determination in Xenopus

In many organisms the proper development of the embryo depends on the asymmetrical distribution of maternal RNAs and proteins in the egg. Although the Xenopus oocyte is radially symmetrical it contains distinct populations of maternal RNAs that are localized either in the animal or vegetal pole. The process of localization of RNAs in Xenopus oocytes occurs during the long period of oocyte differentiation and growth that is accompanied by the elaboration of oocyte polarity. Some of the vegetally localized RNAs, such as Vg1, VegT, and Xwnt11, are involved in axial patterning and germ layer specification. Others, such as Xdazl and Xcat2, which are located in the germ plasm, are likely to play a role in the specification of germ cell fate. We will discuss the different aspects of RNA localization in Xenopus in the context of the differentiation of the germ cells and the development of the oocyte polarity.

The vegetally localized mRNA fatvg is associated with the germ plasm in the early embryo and is later expressed in the fat body.

Vegetally localized RNAs in Xenopus oocytes have been implicated in the establishment of the primary germ layers and the formation and development of the primordial germ cells. fatvg mRNA is localized through the late pathway to the vegetal cortex. Like Vg1 mRNA fatvg is distributed throughout the entire cortex; however, unlike Vg1 there is a small fraction of the fatvg mRNA that is associated with the mitochondrial cloud. In early cleavage stage embryos, fatvg mRNA is associated with the germ plasm located at the tips of the vegetal blastomeres of the embryo. While several localized RNAs that follow the Message Transport Organizer (METRO) pathway have been found in the germ plasm in embryos, fatvg is a late pathway RNA that is associated with the germ plasm. In tadpoles, fatvg mRNA shows a novel pattern of expression which is distinct from the germ cell lineage and is detected at the dorso-anterior margin of the endodermal mass along the midline in two clusters of cells. fatvg mRNA expression is also detected later in the developing fat bodies, the major adipose tissues of the frog.

DEADSouth is a germ plasm specific DEAD-box RNA helicase in Xenopus related to eIF4A

DEADSouth was selected in a screen for localized RNAs in Xenopus oocytes. In situ hybridization analysis shows that DEADSouth localizes to the vegetal cortex via the mitochondrial cloud early in oogenesis and segregates with germ plasm during early embryogenesis. These results lend further support for the general concept that the role of the early RNA localization pathway in Xenopus is to localize germ cell components (reviewed in King, M.L., Zhou, Y., Bubunenko, M., 1999. BioEssays 21, 546–557). Further analysis shows that DEADSouth is a germline specific RNA, found exclusively within the germ plasm of oocytes and PGCs, as well as in male germ cells. Sequence comparisons with DEADSouth show it to be a member of a small sub-family of the DEAD-box RNA-dependent helicases related to eIF4A.

Expression of mRNAs of the Aquaporin Family in Mouse Oocytes and Embryos

The molecular basis of water and cryoprotectant permeability in mammalian oocytes and embryos is poorly understood. Therefore, we investigated the expression of mRNAs of water channel proteins (aquaporins) in mouse oocytes and embryos by RT-PCR. The total RNA of mouse oocytes at metaphase II and embryos at the 4-cell, morula, and blastocyst stages was isolated, reverse-transcribed, and subjected to nested PCR amplification. Aquaporins were expressed in both oocytes and embryos, but the types were different among the developmental stages: aquaporins 3 and 7 were expressed in oocytes and embryos at all stages examined, but aquaporins 8 and 9 were expressed only in blastocysts. On the other hand, aquaporins 1, 2, 4, 5, and 6 were not detected in any of the stages examined. The present study shows for the first time that aquaporins are expressed in mammalian oocytes and embryos. These aquaporins may play a role in water transport and conceivably also in cryoprotectant transport across the plasma membrane in these cells.

Evolutionary conservation of post-transcriptional 3' end adenylation of small RNAs: S. cerevisiae signal recognition particle RNA and U2 small nuclear RNA are post-transcriptionally adenylated.

The 3' terminal nucleotide of several human small RNAs, including Signal Recognition Particle (SRP) RNA, 7SK RNA, U2 small nuclear RNA and ribosomal 5S RNA was previously characterized and a fraction of these RNAs was found to contain a single post-transcriptionally added adenylic acid residue on their 3' ends. Here we report the development of a reverse transcription-polymerase chain reaction (RT-PCR) assay for determining and quantifying the extent of post-transcriptional adenylation of RNAs from different species. Using this assay, we found that a fraction of S. cerevisiae U2 small nuclear RNA and S. cerevisiae SRP RNA contain a post-transcriptionally added adenylic acid residue on their 3' ends. Sequencing analysis confirmed this adenylation to be post-transcriptional. Corresponding small RNAs in Xenopus oocytes also contained this post-transcriptional adenylation on their 3' ends. These data show that post-transcriptional adenylation on the 3' end of several small RNA molecules is conserved through evolution. Xenopus SRP RNA from both cytoplasmic and nuclear compartments contained post-transcriptionally added adenylic acid residue on its 3' end. In addition, the Alu portion of SRP RNA was adenylated, when injected into the cytoplasm of frog oocytes. These data show that this novel adenylating machinery, capable of specifically adding a single adenylic acid to the 3' end of some RNA molecules, is present and functional in both nucleus and cytoplasm.

Relationship between hepatic deiodination of thyroxine and early oocyte development in rainbow trout, Oncorhynchus mykiss

We examined the relationship between five stages of ovarian growth and development and hepatic microsomal thyroxine (T4) deiodinating activity in rainbow trout, Oncorhynchus mykiss, held in the laboratory at 11.5°C. Thyroxine outer-ring deiodination (T4ORD) activity, which forms biologically active 3,5,3'-triiodothyronine (T3), was low when the gonadosomatic index (GSI = (ovary mass X 100) / body mass) was below 0.2 (stage I, previtellogenesis), but rose sharply between GSI values of 0.2 and 0.3 (stage II), when oocyte Balbiani activity, RNA synthesis, and endogenous vitellogenesis were maximal. Hepatic T4ORD activity was again low during exogenous vitellogenesis at GSI values of 0.3-12, which correspond to stages III-V. Hepatic T4 inner-ring deiodination (T4IRD), which forms biologically inactive 3,3',5'-triiodothyronine, was detected only at stage II. Plasma T3 concentrations were significantly higher at stage II than at stage IV (primary yolk globule formation). Our data indicate changes in systemic thyroidal status at stage II and are consistent with a role for T3 in early oogenesis in trout. Enhanced hepatic T3 production at the late Balbiani stage may be required for oocyte development.

Accumulation and localisation of maternal RNA in oocytes of Japanese quail.

Accumulation of total RNA and poly(A+)RNA was determined in the oocytes of Japanese quail (Coturnix coturnix japonica) during oogenesis, by a standard spectrophotometric method, after RNA extraction. Intensive RNA accumulation was observed in the oocytes 0.25-2.0 mm in diameter (correlated with the presence of lampbrush chromosomes), followed by a plateau in 2.0-3.0 mm oocytes (correlated with the condensation of lampbrush chromosomes). Resumption of the RNA accumulation in the 3.0-5.0 mm oocytes is interpreted as a result of external RNA delivery by the granulosa cells. Most of the RNA (approximately 3/4, 3-4 micrograms) contained in the ovulated oocyte was found under the vitelline membrane surrounding the yolk. This RNA has been designated as 'extraembryonic RNA', as it is located outside the germinal disc region where the embryo is formed. The extraembryonic RNA is rapidly degraded within 24 h, from ovulation until oviposition, during egg passage through the oviduct, while the RNA present in a germinal disc (approximately 1.1 micrograms) is stable. In bird oocytes the presence of two, territorially separated pools of RNA has been postulated: one contained in a germinal disc and needed for early embryo development, and the second present in the cytoplasmic layer around the yolk supporting oocyte growth and development during oogenesis.

Cyclin synthesis controls the progression of meiotic maturation in mouse oocytes.

To study the mechanisms involved in the progression of meiotic maturation in the mouse, we used oocytes from two strains of mice, CBA/Kw and KE, which differ greatly in the rate at which they undergo meiotic maturation. CBA/Kw oocytes extrude the first polar body about 7 hours after breakdown of the germinal vesicle (GVBD), whilst the oocytes from KE mice take approximately 3-4 hours longer. In both strains, the kinetics of spindle formation are comparable. While the kinetics of MAP kinase activity are very similar in both strains (although slightly faster in CBA/Kw), the rise of cdc2 kinase activity is very rapid in CBA/Kw oocytes and slow and diphasic in KE oocytes. When protein synthesis is inhibited, the activity of the cdc2 kinase starts to rise but arrests shortly after GVBD with a slightly higher level in CBA/Kw oocytes, which may correspond to the presence of a larger pool of cyclin B1 in prophase CBA/Kw oocytes. After GVBD, the rate of cyclin B1 synthesis is higher in CBA/Kw than in KE oocytes, whilst the overall level of protein synthesis and the amount of messenger RNA coding for cyclin B1 are identical in oocytes from both strains. The injection of cyclin B1 messenger RNA in KE oocytes increased the H1 kinase activity and sped up first polar body extrusion. Finally, analysis of the rate of maturation in hybrids obtained after fusion of nuclear and cytoplasmic fragments of oocytes from both strains suggests that both the germinal vesicle and the cytoplasm contain factor(s) influencing the length of the first meiotic M phase. These results demonstrate that the rate of cyclin B1 synthesis controls the length of the first meiotic M phase and that a nuclear factor able to speed up cyclin B synthesis is present in CBA/Kw oocytes.

Quantitative RT-PCR amplification of RNA in single mouse oocytes and preimplantation embryos.

We describe a simple whole-cell method for quantitative reverse transcription (RT) PCR amplification of RNA that consistently allows the analysis of trace amounts of RNA, such as those carried by a fraction of a single mouse oocyte or preimplantation embryo, without organic extraction. The method is based on a preliminary genomic DNA digestion by DNase I in the presence of Mn++ and a subsequent RT step with rTth Reverse Transcriptase at 70 degrees C with the same buffer components, which also has the effect to irreversibly denature DNase I activity. Because of the completeness of genomic DNA digestion and RNA recovery, this procedure makes it possible to quantitatively amplify any target RNA, including those coded by intronless genes or genes whose intron-exon boundaries are unknown. By taking mRNAs of beta-actin, heat-shock protein HSP70.1 and ribosomal protein S16 as experimental models, we demonstrate the effectiveness of genomic DNA digestion by DNase I-Mn++ and of DNase I heat-denaturation and the quantitative properties of our method. We also show that this procedure is useful for transcriptional analyses during development that are hindered by paucity of biological material.

The role of zinc finger linkers in p43 and TFIIIA binding to 5S rRNA and DNA.

Transcription factor IIIA (TFIIIA) and p43 zinc finger protein form distinct complexes with 5S ribosomal RNA in Xenopus oocytes. Additionally, TFIIIA binds the internal promoter of the 5S RNA gene and supports assembly of a transcription initiation complex. Both proteins have nine tandemly repeated zinc fingers with almost identical linker lengths between corresponding fingers, yet p43 has no detectable affinity for the 5S RNA gene. TFIIIA zinc fingers 1-3 are connected by highly conserved linkers, first identified in the Drosophila protein Krüppel, that are found in many DNA binding zinc finger proteins. To understand the role of these linkers in RNA and DNA binding we exchanged three TFIIIA linker amino acids with the equivalent amino acids from p43. The major effect of linker substitution is a 50-fold reduction in DNA specificity, concomitant with an 8-fold reduction in affinity. N-Terminal zinc fingers from either TFIIIA or p43 bind to multiple specific sites on 5S RNA that are resistant to competition by tRNA or poly(rA). This mode of RNA binding is unaffected by linker substitution. These data suggest that zinc finger linkers significantly facilitate the specificity of DNA binding.

Pharmacological differentiation between neuronal and recombinant glutamate receptor channels expressed in Xenopus oocytes

To determine the molecular components of neuronal glutamate receptors, it is important to identify pharmacological tools that allow differentiation between different glutamate receptor types. Here, we utilized the naphthalene derivative Evans Blue (EB) and a collection of other subtype-specific compounds (polyamine toxins, concanavalin A, cyclothiazide) to compare the pharmacological profile of neuronal and recombinant glutamate receptors GluR1-GluR6 expressed in Xenopus oocytes. Submicromolar concentrations of EB selectively reduced the activity of homomeric glutamate receptors GluR1, GluR2(Q) and GluR4. Applied at concentrations above 100 μM, EB potentiated kainate responses of receptors GluR1, GluR3 and GluR4, while receptors GluR2(Q) and GluR6(Q) were completely blocked. Similar experiments were performed on identified neurones in brain slices and after injection of rat brain RNA in Xenopus oocytes. Neuronal kainate responses were (i) potentiated by 100 μM cyclothiazide, (ii) slightly blocked after preincubation in 10 μM concanavalin A, and (iii) not significantly affected by either low (< 1 μM) or high (>100 μM) concentrations of EB. Their pharmacological properties were markedly different from those of recombinant glutamate receptor channels GluR1-GluR6 investigated in heterologous expression systems.