Influence of nucleotides, cations and nucleoside triphosphatase inhibitors on the release of ribonucleic acid from isolated rat liver nuclei

Agutter, Paul S.

doi:10.1042/bj1880091

Cited by 31 publications

(12 citation statements)

References 35 publications

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“…This model is in agreement with the observation that ATPases are regulated by phosphorylation-dephosphorylation processes (55). Moreover, this model is also in agreement with the findings of Agutter, who has proposed that the dephosphorylation of the nuclear envelope may increase NTPase activity, and that poly(A)-containing RNA (a known activator of NTPase and mRNA effilux) may increase enzyme activity through a dephosphorylation reaction (43). Further, this model is in agreement with the cytochemical studies of Vorbrodt and Maul, who have observed that the NTPase of the nuclear envelope is associated with the nuclear pore complex and mRNA particles translocating through the nuclear pores (51).…”

Section: Discussionsupporting

confidence: 80%

“…RNA transport in vitro involves both intranuclear RNA processing and subsequent efflux, and a source of high-energy phosphate such as ATP is necessary for transport but not processing (32,52,53 (33), and cyclic AMP stimulates both functions whereas NaF inhibits them (32,33,(52)(53)(54). Treatment with trypsin and sulfhydryl blockers inactivates both functions (32,43). Moreover, histochemical studies have suggested that the NTPase resides in the nuclear pore complex (32) and is associated with ribonucleoproteins that are being translocated through the nuclear pore complex (51).…”

Section: Discussionmentioning

confidence: 99%

“…In order to understand the mechanism of mRNA transport (51) from the nucleus, rats have been injected with either radiolabeled orotic acid or uridine and the efflux of labeled RNA from isolated liver nuclei into a surrogate cytoplasm has been studied (32,(52)(53)(54). A number of criteria suggest that the RNA transported under the conditions studied is mRNA [the criteria include size, base and poly(A) content, activity in directing protein synthesis, incorporation into polysomes, and inclusion into specific ribonucleoprotein particles] (32,43,44,52,53). RNA transport in vitro involves both intranuclear RNA processing and subsequent efflux, and a source of high-energy phosphate such as ATP is necessary for transport but not processing (32,52,53 (33), and cyclic AMP stimulates both functions whereas NaF inhibits them (32,33,(52)(53)(54).…”

Section: Discussionmentioning

confidence: 99%

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Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism.

Purrello¹,

Burnham²,

Goldfine³

1983

Proc. Natl. Acad. Sci. U.S.A.

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The direct addition of insulin to highly purified nuclear envelopes prepared from the livers of diabetic rats resulted in a decrease in the incorporation of 32p into trichloroacetic acid-precipitable proteins. Autoradiography of 32P-labeled envelopes, solubilized in sodium dodecyl sulfate and subjected to electrophoresis, revealed that insulin decreased the phosphorylation of all major protein bands. Insulin produced detectable effects at concentrations between 0.1 and 1 pM, maximal effects at 10 pM, and progressively diminished effects at higher concentrations. Two insulin analogs, desdipeptide proinsulin and desoctapeptide insulin, had approximately 10% and 1%, respectively, the activity of native insulin. When nuclear envelopes were first phosphorylated with [y-32P]ATP and insulin was then added with an excess of unlabeled ATP, dephosphorylation was enhanced, suggesting that insulin was regulating nuclear envelope phosphatase activity. The direct addition of insulin to isolated rat liver nuclei in the presence of ATP stimulated the release of previously '4C-labeled trichloroacetic acid-precipitable mRNA-like material, and the direct addition of insulin to nuclear envelopes stimulated the activity of nucleoside triphosphatase, the enzyme that participates in mRNA nucleocytoplasmic transport. Moreover, the dose-response curves for these functions mirrored insulin's inhibition of nuclear envelope phosphorylation. These data suggest, therefore, a mechanism whereby insulin directly inhibits the phosphorylation of the nuclear envelope, leading in turn to the regulation of mRNA metabolism.Insulin has numerous effects on target cells, including regulation of membrane transport, enzyme activities, and DNA and RNA metabolism (1, 2). There is evidence that insulin, like other hormones, regulates a number of cellular functions by phosphorylation and dephosphorylation reactions (3-6). Moreover, effects of insulin on phosphorylation and dephosphorylation have recently been observed in broken cell preparations (7-11).We and others have demonstrated specific high-affinity binding sites for insulin on nuclei and nuclear envelopes of liver and other tissues (12)(13)(14)(15)(16)(17). Moreover, morphological studies have suggested that insulin can enter intact cells and interact with the nuclear envelope (18)(19)(20). However, the exact role of insulin in nuclear functions is unknown. There are a number of reports demonstrating that insulin increases mRNA levels in liver and other tissues (21)(22)(23)(24)(25)(26)(27)(28)(29). Further, it has recently been reported that the direct addition of insulin to isolated nuclei stimulates the release of mRNA (30). These observations suggest, therefore, that one action of insulin on the nucleus is to regulate mRNA metabolism.Several lines of evidence indicate that transport of mRNA from the nucleus is mediated by a specific enzyme of the nuclear envelope, the nuclear envelope nucleoside triphosphatase (NTPase; EC 3.6-. (39). Accordingly, in the present study we have investigated ...

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Section: Discussionsupporting

confidence: 80%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism.

Purrello¹,

Burnham²,

Goldfine³

1983

Proc. Natl. Acad. Sci. U.S.A.

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“…It also indicates that up to 15% of the envelopes are leaky, in accordance with previous experiments (1). Envelope NTPase activity is stimulated by ribonucleic acids, especially by poly(A) (23). As shown in Table 2, this effect is distinct in the leaky DNase envelopes.…”

supporting

confidence: 76%

Permeability measurements with closed vesicles from rat liver nuclear envelopes.

Riedel

Bachmann

Prochnow

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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Closed nuclear envelope ghosts in the physiological orientation were prepared from rat liver nuclei as previously described. Here we report transport measurements of various proteins and ribonucleic acids across the envelope Qf these vesicles. Histones were accumulated rapidly in the ghosts, in contrast to other, nonnuclear, proteins. Triton X-100 removal of the external nuclear membrane from loaded vesicles, as well as comparative studies with open vesicles, excluded the effects of external adsorption. The exchange rate of histones across the nuclear envelope is strongly depressed in the presence of GTP and GDP. The vesicles contain the translocation mechanism for poly(A)-containing RNA. The translocation of poly(A), messenger RNA, and ribosomal RNA was investigated after entrapment of these nucleic acids during the preparation of vesicles. Our data show that the complete export of only poly(A)-containing RNA from the vesicles is enhanced in the presence of 2 mM ATP. This RNA, as well as poly(A), is transported unidirectionally.Previously, we described the preparation and characterization of closed envelopes from rat liver nuclei (1, 2). These vesicles were shown to be tight. The DNA content, calculated per mg of protein, is reduced to 1-1.5% of the value for whole nuclei.The high number of binding sites for nuclear constituents, such as histones or mRNA, renders the exact measurement of rates of migration into or out of whole nuclei difficult. Evidence from microinjection experiments (3,4), sequence analysis, and especially site-directed mutagenesis of proteins that accumulate in the nucleus (5, 6) has prompted the discussion of selectivity of nuclear membrane transport (7). The nuclear envelope ghosts seemed suitable for the further elucidation of these mechanisms.MATERIALS AND METHODS Leaky nuclear envelopes ("DNase envelopes") were prepared by hypotonic DNase I treatment as described by Dwyer and Blobel (8). The preparation of closed nuclear envelopes with heparin solutions was carried out as previously described (1, 2).Poly(adenylic acid) of an average molecular weight of 100,000 was purchased from Sigma, and the absence of poly(A) with molecular weight below 60,000 was verified by HPLC runs on a Nucleogen DEAE 500-10 column. NTPase activity was determined as described (1). Histones were prepared from calf thymus and fractionated by the procedure of Johns (9) (purity control, see Fig. 3 and ref. 10). Xenopus laevis eggs were obtained from the Max-Planck-Institute of Biophysics, Frankfurt, and nucleoplasmin was prepared from these as described (11). All proteins were labeled in 50 mM NaHCO3 by addition of [14C]acetic anhydride (Amersham Buchler), specific activity 120 mCi/mmol (1 Ci = 37 GBq). The proteins were then dialyzed against the buffer described in the legend of Fig. 3 Fig. 1, the closed vesicles still possess residual fringes of the chromatin material on the inside of the inner membrane; the pore complexes are revealed by freeze fracture. Phase-contrast light microscopy reveals a population...

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“…2 shows the kinetics of the release of [3H]RNA from these preparations of nuclei, incubated in the medium described in Materials and Methods, as well as the effects of omitting ATP and its regenerating system, and of substituting the non-hydrolysable P,y-imino analogue of ATP, adenosine 5'-[P,y-imino]triphosphate (AdoPP[NH]P). In other respects the conditions were the same as those used by previous investigators [12,37] with minor variations (the use of creatine kinase and creatine phosphate instead of phosphoenolpyruvate and pyruvate kinase as ATP-regenerating system, and of Escherichia coli RNA instead of yeast RNA as carrier).…”

Section: Maintenance Qf Nuclear Integrity With R N a Release In Vitromentioning

confidence: 99%

Isolation and Purification of Rat Hepatoma Nuclei Active in the Transport of Messenger RNA in vitro

Jacobs

Birnie

1982

Eur J Biochem

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1. A variety of procedures for obtaining metabolically active nuclei from rat hepatoma tissue-culture (HTC) cells has been explored. All gave preparations which were, to a greater or lesser extent, contaminated with cytoplasmic RNA, which appeared to be absorbed non-specifically during the lysis of the cells.2. A medium containing spermidine, monovalent and divalent cations, and cytosol has bccn defined in which the integrity of the isolated nuclei was maintained during incubation in virro.3. The release of steady-state-labelled and pulse-labelled RNA from HTC cell nuclei during incubation in this medium has been studied. The transport of both was dependent on ATP and cytosol, but was distinguishable by the kinetics and by the fact that only the transport of pulse-labelled RNA appeared to involve hydrolysis of ATP. Transported RNA included ribosomal R N A and messenger-likc RNA as judged by criteria of size, oligo(dT)-cellulose binding, and organisation into ribonucleoproteins. A large proportion of the cytoplasmic R N A which adhered to the nuclei was also released during incubation in v i m .4. Although the poly(A)-rich RNA transported in v i m was contaminated with poly(A)-rich RNA sequences arising from non-specific leakage of nuclear RNA and from the release of nucleus-adherent cytoplasmic RNA, the extent of this contamination was comparatively small (10"; and 10-15% by weight respcctively). Thc transportcd poly(A)-rich RNA has been shown to consist of a complex mixture of sequences at heterogeneous abundanccs, resembling polysomal poly(A)-rich mRNA from HTC cells.5. The pattern of relative abundanccs of the sequences in transported poly(A)-rich RNA was intermediate betwieen that of polysomal and nuclear poly(A)-rich RNAs, and indicated that some degree of sequence-spccific selection of processing, or transport, or both, can be maintained in isolated nuclei. Sequence-specific selcction in vitro at the level of individual poly(A)-rich RNA sequences was detected using cloned cDNAs for poly(A)-rich polysomal RNA sequences.

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Influence of nucleotides, cations and nucleoside triphosphatase inhibitors on the release of ribonucleic acid from isolated rat liver nuclei

Cited by 31 publications

References 35 publications

Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism.

Insulin regulation of protein phosphorylation in isolated rat liver nuclear envelopes: potential relationship to mRNA metabolism.

Permeability measurements with closed vesicles from rat liver nuclear envelopes.

Isolation and Purification of Rat Hepatoma Nuclei Active in the Transport of Messenger RNA in vitro

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