The Cleavage and Polyadenylation Specificity Factor in Xenopus laevis Oocytes Is a Cytoplasmic Factor Involved in Regulated Polyadenylation

ABSTRACT During early development, specific mRNAs receive poly(A) in the cytoplasm. This cytoplasmic polyadenylation reaction correlates with, and in some cases causes, translational stimulation. Previously, it was suggested that a factor similar to the multisubunit nuclear cleavage and polyadenylation specificity factor (CPSF) played a role in cytoplasmic polyadenylation. A cDNA encoding a cytoplasmic form of the 100-kDa subunit of Xenopus laevis CPSF has now been isolated. The protein product is 91% identical at the amino acid sequence level to nuclear CPSF isolated from Bos taurusthymus. This report provides three lines of evidence that implicate theX. laevis homologue of the 100-kDa subunit of CPSF in the cytoplasmic polyadenylation reaction. First, the protein is predominantly localized to the cytoplasm of X. laevisoocytes. Second, the 100-kDa subunit of X. laevis CPSF forms a specific complex with RNAs that contain both a cytoplasmic polyadenylation element (CPE) and the polyadenylation element AAUAAA. Third, immunodepletion of the 100-kDa subunit ofX. laevis CPSF reduces CPE-specific polyadenylation in vitro. Further support for a cytoplasmic form of CPSF comes from evidence that a putative homologue of the 30-kDa subunit of nuclear CPSF is also localized to the cytoplasm of X. laevisoocytes. Overexpression of influenza virus NS1 protein, which inhibits nuclear polyadenylation through an interaction with the 30-kDa subunit of nuclear CPSF, prevents cytoplasmic polyadenylation, suggesting that the cytoplasmic X. laevis form of the 30-kDa subunit of CPSF is involved in this reaction. Together, these results indicate that a distinct, cytoplasmic form of CPSF is an integral component of the cytoplasmic polyadenylation machinery.

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A cDNA clone for a polyadenylated RNA-binding protein of Xenopus laevis oocytes hybridizes to four developmentally regulated mRNAs

Molecular and Cellular Biology ◽

10.1128/mcb.5.10.2697-2704.1985 ◽

1985 ◽

Vol 5 (10) ◽

pp. 2697-2704

Author(s):

L J Lorenz ◽

J D Richter

Keyword(s):

Xenopus Laevis ◽

Cdna Clone ◽

Rna Binding ◽

Binding Protein ◽

Single Gene ◽

In Vitro Translation ◽

Xenopus Laevis Oocytes ◽

Cdna Insert ◽

Alternative Processing

Xenopus laevis oocytes contain a unique group of proteins which decrease during oogenesis, bind poly(A) RNA, and possibly play a role in the regulation of translation. A monoclonal antibody generated against one of these proteins was used to screen an expression vector cDNA library. A cDNA clone was isolated and confirmed to code for the binding protein by in vitro translation of hybrid-selected RNA followed by immunoprecipitation. This cDNA, when used in RNA gel blots, hybridized to four transcripts of 2.0, 1.7 (two transcripts of similar size), and 1.2 kilobases. All of the transcripts decreased in amount during oogenesis and were not evident in somatic cells. In addition, the fraction of the transcripts associated with polysomes decreased during oogenesis. Digestion of the cDNA insert with PstI generated two fragments of 220 and 480 base pairs which, when used as probes in an RNA gel blot, hybridized to unique as well as common transcripts. Genomic Southern blots suggested the presence of a single gene, indicating that these transcripts arose by alternative processing.

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Uptake of biotin by native Xenopus laevis oocytes

AJP Cell Physiology ◽

10.1152/ajpcell.1990.259.3.c397 ◽

1990 ◽

Vol 259 (3) ◽

pp. C397-C401 ◽

Cited By ~ 19

Author(s):

H. M. Said ◽

L. Polenzani ◽

S. Khorchid ◽

D. Hollander ◽

R. Miledi

Keyword(s):

Xenopus Laevis ◽

Mammalian Cells ◽

Xenopus Oocytes ◽

Sulfhydryl Group ◽

Maximum Velocity ◽

Significant Inhibition ◽

Metabolic Inhibitors ◽

Xenopus Laevis Oocytes ◽

Uptake System

The present study examined biotin uptake by Xenopus laevis oocytes in vitro. Uptake of low (0.03 microM) and high (10 microM) concentrations of biotin was linear with time for up to 4 h of incubation and occurred with little initial binding to oocytes. Uptake of biotin was dependent on extracellular Na+ concentration [Na+]o and was severely inhibited when Na+ was replaced by other monovalent cations [choline, tetraethylammonia, Li+, and tris(hydroxymethyl)aminomethane]. The initial rate of biotin uptake was saturable as a function of concentration with an apparent Michaelis constant of 3.9 +/- 0.5 microM and maximum velocity of 1,559 +/- 70 fmol.oocyte-1.h-1. Addition to the incubation medium of biotin structural analogues desthiobiotin and thioctic acid caused significant and concentration-dependent inhibition in the uptake of [3H]biotin. This inhibition was found to be competitive in nature with inhibition constant values of 9 and 17.5 microM. In contrast, neither the structural analogue biocytin nor biotin methyl ester (compounds in which the carboxyl group of the valeric acid moiety is blocked) showed any effect on the uptake of [3H]biotin. Biotin uptake was significantly blocked by the metabolic inhibitors dinitrophenol, cyanide, and azide and by incubation at 4 degrees C. Also, the sulfhydryl group blocker p-(chloromercuri)phenylsulfonate caused significant inhibition in biotin uptake. These results demonstrate that Xenopus oocytes possess an uptake system for biotin in its cell membrane that is Na+, energy, and temperature dependent. These characteristics of biotin uptake are similar to those reported in mammalian cells. It is suggested that Xenopus oocytes might be a useful in vitro model system to study the details of the mechanisms and regulation of biotin movement across biological membranes.(ABSTRACT TRUNCATED AT 250 WORDS)

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Remifentanil Directly Activates Human N-methyl-d-aspartate Receptors Expressed in Xenopus laevis Oocytes

Anesthesiology ◽

10.1097/00000542-200406000-00028 ◽

2004 ◽

Vol 100 (6) ◽

pp. 1531-1537 ◽

Cited By ~ 50

Author(s):

Klaus Hahnenkamp ◽

Joke Nollet ◽

Hugo K. Van Aken ◽

Hartmut Buerkle ◽

Tobias Halene ◽

...

Keyword(s):

Nmda Receptor ◽

Postoperative Pain ◽

Xenopus Laevis ◽

Nmda Receptors ◽

Messenger Rna ◽

Xenopus Laevis Oocytes ◽

Opiate Tolerance ◽

Induced Currents ◽

Pore Blocker

Background Clinical studies suggest that intraoperative administration of the clinical remifentanil formulation Ultiva (GlaxoWellcome GmbH & Co, Bad Oldesloe, Germany) increases postoperative pain and postoperative analgesic requirements, but mechanisms remain unclear. N-methyl-D-aspartate (NMDA) receptors are thought to play a major role in development of postoperative pain and opiate tolerance. The authors hypothesized that Ultiva directly stimulates human NMDA receptors. Methods To test this hypothesis, the authors expressed human NR1A/NR2A and NR1A/NR2B NMDA receptors in Xenopus laevis oocytes by injection of messenger RNA prepared in vitro. After protein expression, they used a two-electrode voltage clamp to measure currents induced by NMDA receptor agonists and opioids. Results Noninjected cells were unresponsive to all compounds tested. Glutamate/glycine (1 nM-1 mM each) or Ultiva (0.01 pM-0.1 mM) stimulated NMDA receptors concentration dependently. NR1A/2A EC50 values were 8.0 microM/12 microM for glutamate/glycine and 3.5 nM for Ultiva, and NR1A/2B EC50 values were 3.9 microM/1.9 microM for glutamate/glycine and 0.82 microM for Ultiva. Glycine in combination with Ultiva showed no additive effect compared with Ultiva alone. Ultiva-induced currents were inhibited by MK-801 (pore blocker) but not by 7-CK (glycine antagonist), D-AP5 (glutamate antagonist), or naloxone. Fentanyl (10 microM) did not stimulate NMDA receptors. Conclusion These data indicate that Ultiva but not fentanyl stimulates NMDA receptors of different subunit combinations (NR1A/2A, NR1A/2B). The mechanism seems to be allosteric activation of the NMDA receptor.

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Transcriptional characteristics of in vitro assembled chromatin assayed by microinjection into Xenopus laevis oocytes

FEBS Letters ◽

10.1016/0014-5793(89)80660-x ◽

1989 ◽

Vol 249 (2) ◽

pp. 367-370 ◽

Cited By ~ 2

Author(s):

Herbert Steinbeißer ◽

Ansgar Hofmann ◽

Pierre Oudet ◽

Michael F. Trendelenburg

Keyword(s):

Xenopus Laevis ◽

Xenopus Laevis Oocytes

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In vitro Induction of Germinal Vesicle Breakdown in Xenopus laevis Oocytes by Melittin

Differentiation ◽

10.1111/j.1432-0436.1982.tb01205.x ◽

1982 ◽

Vol 21 (1-3) ◽

pp. 127-132 ◽

Cited By ~ 8

Author(s):

AMRUT K. DESHPANDE ◽

S.S. KOIDE

Keyword(s):

Xenopus Laevis ◽

Germinal Vesicle ◽

Xenopus Laevis Oocytes ◽

Germinal Vesicle Breakdown ◽

In Vitro Induction

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Progesterone-Induced Meiotic Reinitation in vitro in Xenopus laevis Oocytes

Differentiation ◽

10.1111/j.1432-0436.1977.tb01520.x ◽

1977 ◽

Vol 9 (1-3) ◽

pp. 67-76 ◽

Cited By ~ 35

Author(s):

Sabine Schorderet-Slatkine ◽

Michel Schorderet ◽

Etienne-Emile Baulieu

Keyword(s):

Xenopus Laevis ◽

Xenopus Laevis Oocytes

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Nuclear transport of U1 snRNP in somatic cells: differences in signal requirement compared with Xenopus laevis oocytes.

The Journal of Cell Biology ◽

10.1083/jcb.125.5.971 ◽

1994 ◽

Vol 125 (5) ◽

pp. 971-980 ◽

Cited By ~ 27

Author(s):

U Fischer ◽

J Heinrich ◽

K van Zee ◽

E Fanning ◽

R Lührmann

Keyword(s):

Xenopus Laevis ◽

Nuclear Import ◽

Nuclear Transport ◽

Somatic Cells ◽

Xenopus Laevis Oocytes ◽

Xenopus Laevis Oocyte ◽

Nuclear Targeting ◽

Transport Pathway ◽

U1 Snrnp

The signal requirement for the nuclear import of U1 RNA in somatic cells from different species was investigated by microinjection of both digoxygenin-labeled wild type and mutant U1 RNA molecules and in vitro reconstituted U1 snRNPs. U1 RNA was shown to be targeted to the nucleus by a temperature-dependent process that requires the prior assembly of RNPs from the common proteins and the microinjected RNA. Competition in the cell between immunoaffinity-purified U1 snRNPs and digoxygenin-labeled U1 snRNPs reconstituted in vitro showed that the transport is saturable and should therefore be a mediated process. The transport of a karyophilic protein under the same conditions was not affected, indicating the existence of a U snRNP-specific transport pathway in somatic cells, as already seen in the Xenopus laevis oocyte system. Surprisingly, the signal requirement for nuclear transport of U1 snRNP was found to differ between oocytes and somatic cells from mouse, monkey and Xenopus, in that the m3GGpppG-cap is no longer an essential signaling component in somatic cells. However, as shown by investigation of the transport kinetics of m3GpppG- and ApppG-capped U1 snRNPs, the m3GpppG-cap accelerates the rate of U1 snRNP import significantly indicating that it has retained a signaling role for nuclear targeting of U1 snRNP in somatic cells. Moreover, our data strongly suggest that cell specific rather than species specific differences account for the differential m3G-cap requirement in nuclear import of U1 snRNPs.

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Cell cycle dynamics of an M-phase-specific cytoplasmic factor in Xenopus laevis oocytes and eggs.

The Journal of Cell Biology ◽

10.1083/jcb.98.4.1247 ◽

1984 ◽

Vol 98 (4) ◽

pp. 1247-1255 ◽

Cited By ~ 337

Author(s):

J Gerhart ◽

M Wu ◽

M Kirschner

Keyword(s):

Cell Cycle ◽

Xenopus Laevis ◽

Germinal Vesicle ◽

Small Scale ◽

Xenopus Laevis Oocytes ◽

Germinal Vesicle Breakdown ◽

Cytoplasmic Factor ◽

M Phase ◽

Cytostatic Factor ◽

Meiotic Cycle

We have examined the regulation of maturation-promoting factor (MPF) activity in the mitotic and meiotic cell cycles of Xenopus laevis eggs and oocytes. To this end, we developed a method for the small scale extraction of eggs and oocytes and measured MPF activity in extracts by a dilution end point assay. We find that in oocytes, MPF activity appears before germinal vesicle breakdown and then disappears rapidly at the end of the first meiotic cycle. In the second meiotic cycle, MPF reappears before second metaphase, when maturation arrests. Thus, MPF cycling coincides with the abbreviated cycles of meiosis. When oocytes are induced to mature by low levels of injected MPF, cycloheximide does not prevent the appearance of MPF at high levels in the first cycle. This amplification indicates that an MPF precursor is present in the oocyte and activated by posttranslational means, triggered by the low level of injected MPF. Furthermore, MPF disappears approximately on time in such oocytes, indicating that the agent for MPF inactivation is also activated by posttranslational means. However, in the absence of protein synthesis, MPF never reappears in the second meiotic cycle. Upon fertilization or artificial activation of normal eggs, MPF disappears from the cytoplasm within 8 min. For a period thereafter, the inactivating agent remains able to destroy large amounts of MPF injected into the egg. It loses activity just as endogenous MPF appears at prophase of the first mitotic cycle. The repeated reciprocal cycling of MPF and the inactivating agent during cleavage stages is unaffected by colchicine and nocodazole and therefore does not require the effective completion of spindle formation, mitosis, or cytokinesis. However, MPF appearance is blocked by cycloheximide applied before mitosis; and MPF disappearance is blocked by cytostatic factor. In all these respects, MPF and the inactivating agent seem to be tightly linked to, and perhaps participate in, the cell cycle oscillator previously described for cleaving eggs of Xenopus laevis (Hara, K., P. Tydeman, and M. Kirschner, 1980, Proc. Natl. Acad. Sci. USA, 77:462-466).

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The 36-kilodalton embryonic-type cytoplasmic polyadenylation element-binding protein in Xenopus laevis is ElrA, a member of the ELAV family of RNA-binding proteins.

Molecular and Cellular Biology ◽

10.1128/mcb.17.11.6402 ◽

1997 ◽

Vol 17 (11) ◽

pp. 6402-6409 ◽

Cited By ~ 38

Author(s):

L Wu ◽

P J Good ◽

J D Richter

Keyword(s):

Xenopus Laevis ◽

Binding Proteins ◽

Rna Binding ◽

Rna Binding Proteins ◽

Binding Protein ◽

Dominant Negative ◽

Cytoplasmic Polyadenylation ◽

Element Binding Protein

The translational activation of several maternal mRNAs in Xenopus laevis is dependent on cytoplasmic poly(A) elongation. Messages harboring the UUUUUAU-type cytoplasmic polyadenylation element (CPE) in their 3' untranslated regions (UTRs) undergo polyadenylation and translation during oocyte maturation. This CPE is bound by the protein CPEB, which is essential for polyadenylation. mRNAs that have the poly(U)12-27 embryonic-type CPE (eCPE) in their 3' UTRs undergo polyadenylation and translation during the early cleavage and blastula stages. A 36-kDa eCPE-binding protein in oocytes and embryos has been identified by UV cross-linking. We now report that this 36-kDa protein is ElrA, a member of the ELAV family of RNA-binding proteins. The proteins are identical in size, antibody directed against ElrA immunoprecipitates the 36-kDa protein, and the two proteins have the same RNA binding specificity in vitro. C12 and activin receptor mRNAs, both of which contain eCPEs, are detected in immunoprecipitated ElrA-mRNP complexes from eggs and embryos. In addition, this in vivo interaction requires the eCPE. Although a number of experiments failed to define a role for ElrA in cytoplasmic polyadenylation, the expression of a dominant negative ElrA protein in embryos results in an exogastrulation phenotype. The possible functions of ElrA in gastrulation are discussed.

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