Polyadenylated RNA and mRNA export factors in extrachromosomal nuclear domains of vitellogenic oocytes in the yellow mealworm Tenebrio molitor

The nucleus of vitellogenic oocytes of the yellow mealworm Tenebrio molitor contains a karyosphere that consists of condensed chromatin embedded in an extrachromosomal fibrogranular material. Numerous nuclear bodies located freely in the nucleoplasm are also observed. Amongst these bodies, counterparts of nuclear speckles (= interchromatin granule clusters (IGCs)) can be identified by the presence of the marker protein SC35. Microinjections of fluorescently tagged 2??-O-Me(U)₂₂ methyl oligoribonucleotide probes, complementary to poly(A) tails of RNAs, revealed poly(A)⁺ RNA in the vast majority of IGCs. We found that all T. molitor oocyte IGCs contain heterogeneous ribonucleoprotein (hnRNP) core protein A1 localized to IGCs in an RNA-dependent manner. The extrachromosomal material of the karyosphere and some nucleoplasmic IGCs also contain the adapter protein Aly known to provide a link between pre-mRNA splicing and mRNA export. The essential mRNA export factor/receptor NXF1 was colocalized with Aly. In nucleoplasmic IGCs, NXF1 was found to localize in an RNA-dependent manner, whereas it was RNA-independently located in the extrachromosomal material of the karyosphere. We believe our data provide evidence for the implication of nucleoplasmic IGCs in mRNA biogenesis and retention on the path to nuclear export.     

Cell-free translation of messenger RNA from oocytes of Xenopus laevis

The poly(A)⁺ RNA which accumulates during oogenesis in the amphibian Xenopus laevis is shown to be functional mRNA; the RNA was active in the mRNA-dependent “shift assay” for initiation sites in the rabbit reticulocyte lysate, and was an efficient template for protein synthesis in the wheat-germ cell-free system. Analysis of the in vitro protein products showed no differences between the coding properties of poly(A)⁺ RNA extracted from oocytes at all stages of development from previtellogenesis to maturity. In previtellogenic oocytes, the in vitro products of polysomal and of mRNP-associated poly(A)⁺ RNA were also identical. Neither was there any evidence for changes in the coding properties of the poly(A)⁺ mRNA of the oocyte. However, the patterns of oocyte in vivo protein synthesis changed markedly during early vitellogenesis. We conclude that the mRNP-associated poly(A)⁺ RNA present in mature oocytes constitutes the stored maternal mRNA, and that during oogenesis the coding composition of the poly(A)⁺ mRNA synthesised does not change markedly, while some form of translational control operates to direct the changing pattern of protein synthesis.     

Cloning of cDNA sequences derived from poly(A)+ nuclear RNA ofXenopus laevis at different developmental stages: Evidence for stage specific regulation

Summary Nuclear poly(A)⁺ RNA was isolated from gastrula and early tadpole stages ofXenopus laevis, transcribed into cDNA and integrated as double stranded cDNA by the G-C joining method into the Pst cleavage site of plasmid pBR 322. After cloning inE. coli strain HB 101 the clone libraries were hybridized to³²P labelled cDNA derived from nuclear poly(A)⁺ RNA of the two different developmental stages. About 20% of the clones gave a positive hybridization signal thus representing RNA molecules of high and medium abundance. From these clones, some individual clones were identified containing sequences which are not present at the oocyte and gastrula stages but which are transcribed at the early tadpole stage of embryonic development.     

Role of calcium in the localization of maternal poly(A)+RNA and tubulin mRNA in Xenopus oocytes

Summary Poly(A)⁺RNA and tubulin mRNA are localized in the periphery of Xenopus oocytes and become delocalized during meiotic maturation. Delocalization of this RNA can be triggered by incubation in agents which reduce entry of calcium ions into the cell (e.g. lanthanum chloride and verapamil). Although these agents ordinarily promote meiotic maturation, addition of theophylline to the medium will inhibit maturation but not delocalization. Manipulations which prevent calcium entry without inducing meiotic maturation (e.g. calcium-free buffer) are also shown to trigger disruption of the RNA localization. In addition, manipulations which reduce chloride efflux from the cell (e.g. increasing the external chloride ion concentration with choline chloride) result in disruption of the localization of poly (A)⁺ RNA and tubulin mRNA without inducing meiotic maturation. The calcium-dependent chloride efflux present in Xenopus oocytes disappears after the oocyte has been stimulated to proceed through meiotic maturation. We show that reduction of the influx of calcium ions or efflux of chloride ions induces the delocalization of poly (A)⁺RNA and tubulin mRNA without inducing meiotic maturation. We suggest, therefore, that reducing the transmembrane movement of these ions is likely to be the natural trigger for the delocalization of poly(A)⁺RNA and tubulin mRNA.     

A Screening Method Tuned for mRNA Processing Factors in Human Cells by Evaluation of the Luciferase Reporter Activity and the Subcellular Distribution of Bulk Poly(A)+ RNA

Screening of mRNA export factors in Saccharomyces cerevisiae and Drosophila melanogaster has identified a number of mRNA processing factors involved in multiple mRNA processing steps. However, only limited information is available on human cells. Here we established a screening system searching for mRNA processing factors in human cells by combining the luciferase reporter system and fluorescence in situ hybridization, which evaluates the nuclear/cytoplasmic distribution of bulk poly(A)⁺ RNA. This system makes it possible to search for the compounds affecting mRNA processing from the various resources.     

Differential accumulation of two size classes of poly(A) associated with messenger RNA during oogenesis in Xenopus laevis

The RNA of full-grown oocytes of Xenopus laevis contains two distinct size classes of poly(A), designated poly(A)_S and poly(A)_L, which contain 15–30 (mean = 20) and 40–80 (mean = 61) A residues, respectively. Both poly(A)_L and poly(A)_S are associated with RNA which is heterogeneous in size. The two classes of poly(A)⁺ RNA can be separated by affinity chromatography: Only poly(A)_L⁺ RNA binds to oligo(dT)-cellulose under appropriate conditions, but up to 50% of the poly(A)_S⁺ RNA can be isolated from the void fraction by binding to poly(U)-Sepharose. Both classes of poly(A)⁺ RNA are active as messenger RNA in an in vitro system and yield identical patterns of in vitro protein products. Previtellogenic oocytes contain almost exclusively poly(A)_L, which accumulates up to vitellogenesis but remains almost constant in amount (molecules/oocyte) during vitellogenesis and in the full-grown oocyte. Poly(A)_S accumulates (molecules/oocyte) from early vitellogenesis up to the full-grown oocyte. The total number of poly(A)⁺ RNA molecules per oocyte increases throughout oogenesis from 2 × 10¹⁰/previtellogenic oocyte [80–90% poly(A)_L] to 20 × 10¹⁰/full-grown oocyte (25–40% poly(A)_L). It is argued that poly(A)_S is protected from degradation in the oocyte, thus stabilizing the “maternal” poly(A)⁺ mRNA.     

Analysis of the membrane-associated poly(A)+RNA in the cytoplasm of dormant Artemia cysts by DNA excess hybridization: Evidence for a nuclear origin

Encysted embryos of Artemia contain latent mRNA, to a large extent associated with a fraction of cytoplasmic membranes. The membranes, purified by EDTA treatment and banding in a sucrose gradient at 1.17 g/cm³, include endoplasmic vesicles and mitochondria. The origin of the membrane-associated poly(A)⁺RNA was therefore investigated. In gel electrophoresis poly(A)⁺RNA from the purified membranes of dormant cysts forms two distinct bands at approx. 3·10⁵ and 5·10⁵ Da. Later during development the lighter component decreases. Nuclei from dormant cysts are devoid of poly(A)⁺RNA, while nuclei from developing embryos (50% emergence) contain a predominant poly(A)⁺RNA component of approx. 5·10⁵ Da. ¹²⁵I-labelled preparations of nuclear DNA and of nuclear and membrane-associated poly(A)⁺RNA were used in reassociation and hybridization experiments with excess nuclear DNA. Poly(A)⁺RNA from the membranes of dormant cysts hybridized to nuclear DNA to the same extent as the nuclear poly(A)⁺RNA from developing embryos. The hybridization of labelled, nuclear poly(A)⁺RNA to nuclear DNA was strongly inhibited by unlabelled membrane RNA from either dormant cysts or developing embryos. It is concluded that the stored, membrane-associated poly(A)⁺RNA in dormant cysts is essentially of nuclear origin. The 5·10⁵-Da component is largely homologous with the corresponding component of nuclear poly(A)⁺RNA at later stages.     

A comparison of sequence complexity of nuclear and polysomal poly(A)+ RNA from different developmental stages ofXenopus laevis

Summary Nuclear poly(A)⁺ and polysomal poly(A)⁺ RNA were isolated from gastrula and early tadpole stages of the amphibianXenopus laevis. Complementary DNA was synthesized from all RNA preparations. Hybridization reactions revealed that at least all abundant and probably most of the less frequent nuclear and polysomal poly(A)⁺ RNA species present at the gastrula stage are also present at the early tadpole stage. On the other hand, there are nuclear RNA sequences at the latter stage which appear, if at all, only at lower concentrations at the gastrula stage. The polysomal poly(A)⁺ RNA hybridization reactions suggest the existence of polysomal poly(A)⁺ RNA sequences at early tadpole stages which are not present in the corresponding gastrula stage RNA.By cDNA hybridization with poly(A)^– RNA it could be shown that most of the poly(A)⁺ containing RNA sequences transcribed into cDNA were also present within the poly(A)^– RNA. It was estimated, that these sequences are 10 fold more abundant within the poly(A)^– polysomal RNA and 3–6 more abundant within the poly(A)^– nuclear RNA as compared to the poly(A)⁺ RNAs.     

NTF2-like domain of Tap plays a critical role in cargo mRNA recognition and export

Metazoan Tap-p15 (also called Nxf1-Nxt1) and yeast Mex67-Mtr2 heterodimers are the general mRNA export receptors. The RNA binding activity of Tap-p15, which is essential for mRNA nuclear export, has been attributed to the amino-terminal RNA binding module of Tap consists of RNA recognition motif (RRM) and leucine-rich repeat. In this study, we identified a novel RNA interaction surface in the NTF2-like (NTF2L) domain of Tap, which is analogous to the rRNA binding platform of Mex67-Mtr2. Tap-p15 uses the three domains to tightly bind the retroviral constitutive transport element. The RNA binding through the NTF2L domain is functionally relevant as introduction of mutations in this region reduced CTE-containing mRNA export activity. In contrast, only when the RRM and NTF2L domains were mutated simultaneously, bulk poly (A)⁺ RNA export and in vivo poly (A)⁺ RNA binding activities of Tap-p15 were significantly attenuated. Moreover, an engineered human cell line harboring the NTF2L domain mutation in the NXF1 gene showed a synthetic growth phenotype and severe mRNA export defect under Aly/REF and Thoc5 depleted condition. These data suggest that Tap-p15 recognizes bulk mRNAs through combinatorial use of the distinct RNA binding domains.     

Nup211, the fission yeast homolog of Mlp1/Tpr,is involved in mRNA export

Synthetic lethal mutants have been previously isolated in fission yeast Schizosaccharomyces pombe, which genetically interact with spmex67, in order to identify the genes involved in mRNA export. The nup211 gene was isolated by complementation of the growth defect in one of the synthetic lethal mutants, SLMex2, under synthetic lethal condition. We showed that Nup211, fission yeast homolog of Mlpl/Mlp2/Tpr, is essential for vegetative growth and Nup211-GFP proteins expressed at endogenous level are localized mainly in nuclear periphery. The accumulation of poly(A)⁺ RNA in the nucleus is exhibited when expression of nup211 is repressed or over-expressed. These results suggest that the Nup211 protein plays a pivotal role of mRNA export in fission yeast.     

The metabolism of a poly(A) minus mRNA fraction in HeLa cells

About 30% of HeLa cell mRNA lacks poly(A) when labeled in the presence of different rRNA inhibitors. Our method of RNA fractionation precludes contamination of the poly(A)^? mRNA with large amounts of poly(A)⁺ sequences. The poly(A)^? species is associated with polyribosomes, has an average sedimentation value equal to or greater than poly(A)⁺ mRNA, and behaves like the poly(A)⁺ mRNA in its sensitivity to EDTA and puromycin release from polyribosomes. There is very little, if any, hybridization at Rot values characteristic of abundant RNA sequences between the poly(A)^? RNA fractions from total cytoplasm or from polyribosomes and ³H-cDNA made to poly(A)⁺ RNA. This indicates that poly(A)^? mRNA does not arise from poly(A)⁺ mRNA by nonadenylation, deadenylation, or degradation of random abundant mRNA sequences. The rate of accumulation of poly(A)^? mRNA larger than 9S in the cytoplasm parallels the accumulation of poly(A)^? mRNA. The poly(A)^? mRNA is maintained as approximately 30% of the total labeled mRNA in a short (90 min) and in a long (20 hr) time period. These data indicate that poly(A)^? mRNA is not short-lived nuclear or cytoplasmic heterogeneous RNA contamination, and that the half-life of the poly(A)^? mRNA may parallel that of the poly(A)⁺ mRNA. Cordycepin appears to almost completely (95%) inhibit poly(A)⁺ mRNA while only partially (60%) inhibiting the poly(A)^? mRNA. The origin of the cordycepin-insensitive mRNA has not been ascertained.     

Isolation of synthetic lethal mutations in combination with spnab2 of fission yeast

spNab2 is a fission yeast, Schizosaccharomyces pombe, homologue of the budding yeast Nab2 protein that is an essential poly(A)⁺ RNA-binding protein required for both nuclear export of mRNA to cytoplasm and poly(A)⁺ tail length control. Here we performed a synthetic lethal genetic screen in the fission yeast to isolate mutants that are genetically linked to spnab2. We isolated three mutants that showed synthetic lethality under the repressed condition of the spnab2 expression. These mutants defined in different complementation groups. All the mutants exhibited the accumulation of poly(A)⁺ RNA in the nucleus under the restricted condition. In addition, the growth defects of one mutant (SLnab2) were complemented partially by some genes (mlo3 and rae1) required for mRNA export, while those of the rest (SLnab1 and SLnab3) were not complemented by any S. pombe genes we tested, which were known to be involved in mRNA export. These results suggest that the isolated mutants might harbor mutations in novel genes functionally linked to the spnab2 gene.     

Characterization of Rat8 localization and mRNA export in Saccharomyces cerevisiae during the brewing of Japanese sake

Ethanol affects the nuclear export of mRNA in a similar way to heat shock in Saccharomyces cerevisiae. We recently reported that the nuclear accumulation of Rat8 caused by ethanol stress correlates well with blocking of the export of bulk poly(A)⁺ mRNA. Here, we characterize the localization of Rat8 and bulk poly(A)⁺ mRNA in sake (Japanese rice wine) yeast during the brewing of sake. In wine must and synthetic dextrose medium, sake yeast showed the same responses to ethanol regarding changes in the localization of Rat8 as wine yeast and a laboratory strain: i.e., cells began the nuclear accumulation of Rat8 at an ethanol concentration of 6% and completed it at 9%. In contrast, during the sake-brewing process, sake yeast showed unique phenomena: i.e., cells did not start the nuclear accumulation of Rat8 until the ethanol concentration of the sake mash reached around 12% and they showed a normal localization of Rat8 around the nuclear envelope at the late stage of fermentation. These results provide new information about the transport of mRNA in yeast cells during actual alcoholic fermentation.     

Isolation of a novel rmn1 gene genetically linked to spnab2 with respect to mRNA export in fission yeast

In fission yeast, Schizosaccharomyces pombe, the spnab2 gene encodes an ortholog of the budding yeast nuclear abundant poly(A)⁺ RNA-binding protein 2 (Nab2) that is an essential protein required for both mRNA biogenesis and nuclear export of mRNA to the cytoplasm. We have previously isolated three mutants (SLnab1–3) that showed synthetic lethality under the repressed condition of spnab2 expression. In this study, we isolated a novel rmn1 gene as a multicopy suppressor that complemented the defects in growth and mRNA export of SLnab1 mutant cells. The rmn1 gene contained three introns and encoded a 589 amino-acid protein with the RNA recognition motif (RRM) in the central region. The Δrmn1 null mutant was viable but showed a s light mRNA export defect. However, its over-expression caused a deleterious effect on growth accompanied by intense accumulation of poly(A)⁺ RNA in the nucleus. The combination of Δrmn1 with Δspnab2 or Δspmex67 also inhibited growth. In addition, Rmn1p was associated with Rae1p in vivo. These results suggest that rmn1 is a novel gene that is functionally linked to spnab2.