Intracellular transport of nuclear ribosomal RNA in Acetabularia mediterranea]

The ribosomal RNA transport from a nucleus to a perinuclear cytoplasm and its following distribution in the cytoplasm of Acetabularia mediterranea cells were studied using transplantation of RNA-labeled rhizoid into unlabeled stalk. In addition rifamycin treatment was used for inhibition of cytoplasmic RNA synthesis. Acetabularia nuclei contain the stable RNA fractions similar to those present in some other eukaryotes. Nuclear 25S and 17S ribosomal RNA rapidly enter the rhizoid cytoplasm whereas the following trasfer of them to other regions of the cell is a very slow process. Within two days only an insignificant part of 25S and 17S ribosomal RNA is transferred from the rhizoid to the stalk and is distributed there over the base-apical gradient. No preferential transfer of the nuclear ribosomal RNA to the apical region was observed.     

Chicken erythrocyte membranes: comparison of nuclear and plasma membranes from adults and embryos

These experiments were designed to determine whether the migration of RNA molecules from an implanted nucleus to the host cytoplasm and from there into the host cell nucleus against a concentration gradient might reflect an artefact induced by the process of nuclear transplantation. That is, are RNA molecules, as previously shown for certain nuclear proteins, caused to artefactually leave a manipulated nucleus and then move into the host cell nucleus (as well as return to the grafted nucleus) during the recovery period?A variety of experiments involving different kinds of manipulative sequences and different numbers of nuclear transplantations suggest—but do not prove—that no artefact is involved in the migration of RNA from one nucleus to another but two experiments strongly support the view that the shuttling activity is a normal physiological process. One of the latter involved a determination of the rate of egress of ³H-RNA from an implanted nucleus and reveals that that rate, in contrast with the equivalent rate of egress for labeled proteins which is clearly abnormal after micromanipulation, is totally consonant with the rate of movement of RNA from nucleus to cytoplasm established from experiments that do not involve micromanipulation. The other experiment involves comparison of (1) the amount of radioactivity acquired by an unlabeled nucleus present in the cell at the time a labeled nucleus is implanted with (2) the amount of radioactivity acquired by an unlabeled nucleus implanted after a labeled nucleus had been implanted and had time to recover from any possible operation-induced trauma. With ³H-protein nuclei the host nuclei of (1) acquired much more label than the host nuclei of (2) because in (1) the host nuclei were able to acquire much of the artefactually-released ³H-protein. For the ³H-RNA experiments, however, little difference was found between (1) and (2) in the amount of label acquired by the host cell nuclei. It can be concluded that little, if any, of the non-random shuttling activity of RNA molecules can be a reflection of an artefact.     

Stable nuclear RNA returns to post-division nuclei following release to the cytoplasm during mitosis

When nuclei from ³H-RNA-containing amebae (A. proteus), chased for as many as 8 cell generations, are implanted into unlabeled enucleate cells, the nuclei retain 30% or more of the cellular ³H-RNA (or at least 15 times the cytoplasmic concentration of ³H-RNA). After such cells divide, the daughter nuclei retain approximately the same proportion of total cellular ³H-RNA—although all (or almost all) of the nuclear RNA is liberated to the cytoplasm during mitosis. Thus, we conclude that RNA stably associated with the interphase nucleus has a particular affinity for the nucleus despite the fact it is in the cytoplasm when the chromosomes are condensed and the nuclear envelope is not intact.     

Polar distribution of RNA in the cytoplasm of Acetabularia mediterranea]

The distribution of total RNA and its individual fractions in two regions of Acetabularia mediterranea stem during regeneration was investigated. During regeneration of both the nuclear and enucleated cells, RNA concentration increases in the cytoplasm of growth zone whereas it changes insignificantly in the central stem region. A study of the qualitative RNA composition in the same stem regions has shown that during regeneration high molecular weight RNA fractions (main peaks - 0,56-10(6) and 1.04-10(6) Dalton) are found in the growth zone and are practically absent from the central cell region. Low molecular weight RNA (supposedly, tRNA and products of RNA destruction) are present in both the cell regions under study.     

Proteins in nucleocytoplasmic interactions. 3. Redistributions of nuclear proteins during and following mitosis in Amoeba proteus

The behavior of nuclear proteins in Amoeba proteus was studied by tritiated amino acid labeling, nuclear transplantation, and cytoplasmic amputation. During prophase at least 77% (but probably over 95%) of the nuclear proteins is released to the cytoplasm. These same proteins return to the nucleus within the first 3 hr of interphase. When cytoplasm is amputated from an ameba in mitosis (shen the nuclear proteins are in the cytoplasm), the resultant daughter nuclei are depleted in the labeled nuclear proteins. The degree of depletion is less than proportional to the amount of cytoplasm removed because a portion of rapidly migrating protein (a nuclear protein that is normally shuttling between nucleus and cytoplasm and is thus also present in the cytoplasm) which would normally remain in the cytoplasm is taken up by the reconstituting daughter nuclei. Cytoplasmic fragments cut from mitotic cells are enriched in both major classes of nuclear proteins, i.e. rapidly migrating protein and slow turn-over protein. An interphase nucleus implanted into such an enucleated cell acquires from the cytoplasm essentially all of the excess nuclear proteins of both classes. The data indicate that there is a lack of binding sites in the cytoplasm for the rapidly migrating nuclear protein. The quantitative aspects of the distribution of rapidly migrating protein between the nucleus and the cytoplasm indicate that the distribution is governed primarily by factors within the nucleus.     

Transport of different RNA species from the nucleus to the cytoplasm in Xenopus laevis neurula cells

Isolated cells from Xenopus laevis neurulae were labeled, and the RNAs extracted from their nuclear and soluble cytoplasmic fractions were analyzed on polyacrylamide gels. In the soluble cytoplasm, 4S RNA emerged very rapidly, and this was immediately followed by the emergence of poly(A)-containing RNA and 18S ribosomal RNA. In contrast, the emergence of 28S ribosomal RNA was delayed by about 2 hr. The size distribution of cytoplasmic poly(A)-containing RNA was much smaller as compared to that of nuclear poly(A)-containing RNA. These results indicate that the newly synthesized RNAs in Xenopus neurula cells are transported from the nucleus to the cytoplasm in a characteristic sequence.     

Autoradiographic studies on the distribution of labeled maternal RNA in early mouse embryos

Maternal RNA of mouse eggs and embryos was labeled by exposure of growing ovarian oocytes to 3H-uridine in vivo 8 to 16 days before ovulation and fertilization. Labeled embryos from the 1-cell stage to the blastocyst stage were collected, fixed, and autoradiographs of plastic sections prepared. The observed grain density was similar in the pronuclei and in the cytoplasm of 1-cell embryos. Knowing the volumes of nucleus and cytoplasm, it was determined that 3% of the maternal RNA was found in the pronuclei. It is suggested that some of this nuclear RNA may be stable small nuclear RNAs (e.g. U1 RNA) retained from the germinal vesicle stage through meiotic maturation. During the 2-cell stage and beyond, maternal RNA is degraded and labeled precursor is reincorporated into nuclear RNA, making it difficult to accurately quantitate the amount of nuclear maternal RNA. It is known that about one third of the total maternal RNA is lost between the 8-cell and blastocyst stages. It was found that cytoplasmic grain densities in inner and outer cells of the morula and blastocyst were not significantly different. Thus, the loss of maternal RNA does not proceed more rapidly in the differentiating trophoblast than in the inner cell mass.     

Characterization of RNA molecules restricted to the nucleus in mouse L-cells

RNA molecules which are restricted to the nucleus in mouse L-cells were characterized by the technique of RNA/DNA hybridization. Competition of cytoplasmic RNA with labeled nuclear RNA of various sizes revealed that the RNA restricted to the cell nucleus is heterogeneous in size. Competition for sites on fractions of mouse DNA of various base compositions indicated that this unstable RNA is also heterogeneous in base composition. Fractionation of nuclei into three subfractions failed to separate the uniquely nuclear RNA from the precursors of cytoplasmic RNA. The significance of the selective transport of RNA from the nucleus to the cytoplasm and its importance in the control of gene activity in eucaryotic cells is discussed.     

Newly formed mRNA lacking polyadenylic acid enters the cytoplasm and the polyribosomes but has a shorter half-life in the absence of polyadenylic acid.

Labeled adenovirus type 2 nuclear RNA molecules from cells treated with 3'-deoxyadenosine (3'dA) were earlier reported to lack polyadenylic acid [poly(A)], but to be correctly spliced in the nucleus (M. Zeevi et al., Cell 26:39-46, 1981). We have now found that the shortened mRNA molecules, lacking poly(A), can also be found in the cytoplasm of 3'dA-treated cells in association with the polyribosomes. In addition, the accumulation of labeled, nuclear adenovirus-specific RNA complementary to early regions 1a, 1b, and 2 of the adenovirus genome was approximately equal in 3'dA-treated and control cells. At the initial appearance of newly labeled adenovirus type 2 RNA (10 min) in the cytoplasm, there was one-half as much labeled RNA in 3'dA-treated cells as in the control. However, control cells accumulated additional mRNA in the cytoplasm very rapidly in the first 40 min of labeling, whereas the 3'dA-treated cells did not. Therefore, it appears that the correctly spliced, poly(A)- mRNA molecules that are labeled in the presence of 3'dA can be transported from the nucleus with nearly the same frequency and the same exit time as in control cells and can be translated in the cytoplasm but have a much shorter half-life than the poly(A)+ mRNA molecules from control infected cells. From these results it is suggested that the role of poly(A) may be entirely to increase the longevity of cytoplasmic mRNA.     

Light-regulated nuclear localization of phytochromes

Phytochrome is a soluble protein that regulates various responses of plants to light. Not all but most of the phytochrome responses are accompanied by changes in the pattern of gene expression. Upon light activation, phytochrome is imported into the nucleus by the nuclear localization activity of the carboxy-terminal half of the molecule. In darkness, the amino-terminal chromophoric domain suppresses this activity to retain the molecule in the cytoplasm. In the nucleus, light-activated phytochrome forms speckles whose biological function remains unclear.     

NUCLEOLAR-DERIVED RIBONUCLEIC ACID IN CHROMOSOMES, NUCLEAR SAP, AND CYTOPLASM OF CHIRONOMUS TENTANS SALIVARY GLAND CELLS

The distribution of monodisperse high molecular weight RNA (38, 30, 28, 23, and 18S RNA) was studied in the salivary gland cells of Chironomus tentans. RNA labeled in vitro and in vivo with tritiated cytidine and uridine was isolated from microdissected nucleoli, chromosomes, nuclear sap, and cytoplasm and analyzed by electrophoresis on agarose-acrylamide composite gels. As shown earlier, the nucleoli contain labeled 38, 30, and 23S RNA. In the chromosomes, labeled 18S RNA was found in addition to the 30 and 23S RNA previously reported. The nuclear sap contains labeled 30 and 18S RNA, and the cytoplasm labeled 28 and 18S RNA. On the basis of the present and earlier analyses, it was concluded that the chromosomal monodisperse high molecular weight RNA fractions (a) show a genuine chromosomal localization and are not due to unspecific contamination, (b) are not artefacts caused by in vitro conditions, but are present also in vivo, and (c) are very likely related to nucleolar and cytoplasmic (pre)ribosomal RNA. The 30 and 23S RNA components are likely to be precursors to 28 and 18S ribosomal RNA. The order of appearance of the monodisperse high molecular weight RNA fractions in the nucleus is in turn and order: (a) nucleolus, (b) chromosomes, and (c) nuclear sap. Since both 23 and 18S RNA are present in the chromosomes, the conversion to 18S RNA may take place there. On the other hand, 30S RNA is only found in the nucleus while 28S RNA can only be detected in the cytoplasm, suggesting that this conversion takes place in connection with the exit of the molecule from the nucleus.     

YAP nuclear‐cytoplasmic translocation is regulated by mechanical signaling,protein modification,and metabolism

Nuclear‐cytoplasmic transport is necessary for the biological function of nuclear proteins. The mechanism underlying this process is very complex and has been a subject of intense research. Yes‐associated protein (YAP), a Hippo signaling pathway effector, localizes to both the cytoplasm and the nucleus and can influence cell proliferation, stem cell status, and tissue homeostasis. Recent studies have focused on the significance of YAP distribution between the nucleus and the cytoplasm in disease, but it remains unclear how this dynamic process is regulated. In this review, we discuss YAP nuclear‐cytoplasmic transport under different physiological and pathological conditions in terms of mechanical signaling, protein modification, and metabolism. Understanding the mechanisms underlying nuclear‐cytoplasmic YAP transport mechanism under different physiological and pathological conditions may help identify important targets for disease treatment.     

The transcript from a DNA puff of Rhynchosciara and its migration to the cytoplasm.

Electrophoretic analysis of 3H-RNA obtained from the proximal sections of Rhynchosciara salivary glands at two distinct developmental periods, one characterized by the presence and the other by the absence of the giant B-2 DNA puff, revealed that the appearance of a 14S poly(A)+ RNA is correlated with the opening of this puff. That this RNA species is transcribed from this puff is indicated by the fact that it is found in RNA extracted from B-2 puffs obtained by microdissection. This confirmed by the specific hybridization of the 14S poly(A)+ RNA to the B-2 locus. Our data indicate that the polyadenylation process takes place at the chromosome level, and that the nuclear sap is not an important compartment in the transport of polyadenylated RNA from the chromosome to the cytoplasm. The kinetics of migration of the 14S species to the cytoplasm were studied; the data indicate that this process is very rapid and, in addition, that the 14S RNA is unstable.     

Gradient of RNA distribution in the cytoplasm of Acetabularia mediterranea

Radiochemical studies and electrophoresis showed that there exists an apico-basal gradient of RNA concentration in Acetabularia mediterranea cytoplasm. The main contribution to the formation of such a gradient is made by the different rates of RNA turnover in the cytoplasm rather than by the transfer of nuclear RNA. High molecular weight RNA fractions synthesized in the cytoplasm originate from chloroplast ribosomes; their sedimentation constants are close to those of 23S and 16S rRNA fractions of E. coli.     

A cell-free system for light-dependent nuclear import of phytochrome

Translocation from the cytosol to the nucleus is an essential step in phytochrome (phy) signal transduction. In the case of phytochrome A (phyA), this step occurs with the help of FHY1 (far-red-elongated hypocotyl 1), a specific transport protein. To investigate the components involved in phyA transport, we used a cell-free system that facilitates the controlled addition of transport factors. For this purpose, we isolated nuclei from the unicellular green algae Acetabularia acetabulum . These nuclei are up to 100 μm in diameter and allow easy detection of imported proteins. Experiments with isolated nuclei of Acetabularia showed that FHY1 is sufficient for phyA transport. The reconstituted system demonstrates all the characteristics of phytochrome transport in Arabidopsis thaliana . In addition, FHY1 was also actively exported from the nucleus, consistent with its role as a shuttle protein in plants. Therefore, we believe that isolated Acetabularia nuclei may be used as a general tool to study nuclear transport of plant proteins.