Covalent attachment of ribonucleic acids to proteins

As a prerequisite for the synthesis of affinity labels, we describe methods to couple histones to ribonucleic acids. For the synthesis of these covalent hybrid molecules, we used a population of histones H1, H2A, H2B, H3, and H4 from calf thymus and polyadenylic acid with an average chain length of up to 260–280 bases, representing the size of poly(A)-tails from mature mRNAs. Three methods were investigated. (a) Poly(A) containing an 8-N₃-A residue was cross-linked to histones by ultraviolet irradiation. (b) The 3-end of the polynucleotide was connected to a mononucleotide containing an aliphatic amino group, and the resulting poly(A)-derivative was coupled to histones via derivation with a bromoacetyl group. (c) The 3-end of the polynucleotide was oxidized with sodium periodate and bound covalently to an amino group of the polypeptide. To demonstrate the RNA content of the hybrid molecule, the poly(A) was removed with RNase T₂.     

Poly(A) binding proteins located at the inner surface of resealed nuclear envelopes.

We have used a photoreactive cross-linking reagent, poly(A/8-N3-A) (a poly(A) of average molecular mass of 100 kDa in which 5-10% of the A residues are replaced by 8-N3-A), to label poly(A) binding proteins of rat liver nuclear envelopes. This reagent was prepared by polymerizing a mixture of ADP and 8-N3-ADP with polynucleotide phosphorylase. The purified poly(A) was labeled in the 5'-position with a 32P group. In nuclear envelopes prepared by a low salt DNase I procedure, the poly(A/8-N3-A) labeled a protein-nucleic acid complex of approximately 270 kDa, which on degradation with RNase U2 or NaOH at pH 10 yielded two polypeptides of approximately 50 and 30 kDa. These photoreaction products were markedly decreased when resealed nuclear envelopes or non-nuclear envelope proteins were irradiated in the presence of poly(A/8-N3-A). The affinity labeling was intensified when resealed vesicles were made leaky by freezing or ultrasonication, suggesting that the poly(A) binding proteins are accessible from the nucleoplasmic but not the cytoplasmic face of the envelope. Moreover binding was specific for poly(A). Alternative reagents, random poly(A/8-N3-A,C,G,U) of about 100 kDa and poly(dA) (molecular mass between 350 and 515 kDa), showed a very low affinity for poly(A) recognition proteins in the low salt DNase I-treated nuclear envelopes; the 270-kDa band was labeled only weakly. The binding site was not protected by poly(A,C,G,U), weakly by poly(dA), and distinctly by poly(A).     

On the existence of polyadenylated histone mRNA in Xenopus laevis oocytes

In a variety of systems, histone mRNA has been shown to lack poly(A) (Adesnik and Darnell, 1972;Grunstein et al., 1973). We have found, however, that in Xenopus laevis oocytes, poly(A)-containing mRNA codes for histones, in a wheat germ cell-free system, based on the following criteria: first, co-migration with authentic X. laevis oocyte histones on polyacrylamide gels; second, no detectable incorporation of tryptophan; third, differential incorporation of lysine and methionine into histone fraction H2A; fourth, resistance of histone fraction H2A to cleavage with cyanogen bromide; and fifth, correspondence of tryptic peptide maps of partially purified cell-free products with authentic X. laevis oocyte histone. RNA which directs the synthesis of histones in the cell-free system is retained on oligo(dT)-cellulose, even after denaturation in 80% DMSO at 70°C, thereby demonstrating the covalent attachment of polyadenylic acid sequences to the mRNA. Poly(A)^? RNA (7S–14S fraction) was also found to code for histones using the same criteria. We discuss the significance of the finding that X. laevis oocytes contain two classes of histone mRNA as well as the potential developmental implications of this observation.     

Evidence for a direct interaction of Rev protein with nuclear envelop mRNA-translocation system.

The interaction of the Rev protein from human immunodeficiency virus type 1 (HIV-1) with the nucleocytoplasmic mRNA-transport system was investigated. In gel-shift assay, the recombinant Rev protein used in this study selectively bound to the Rev-responsive element (RRE) region of HIV-1 env-specific RNA. Nitrocellulose-filter-binding studies and Northern/Western-blotting experiments revealed an association constant of approximately 1 x 10(10) M-1. The Rev protein also strongly bound to isolated nuclear envelopes from H9 cells, containing the poly(A)-binding site (= mRNA carrier) and the nucleoside triphosphatase (= NTPase), which are thought to be involved in nuclear export of poly(A)-rich mRNA. Binding of 125I-Rev to a 110-kDa nuclear-envelope protein, the putative mRNA carrier, could be demonstrated in in vitro experiments. Both efflux of cellular poly(A)-rich RNA, such as actin RNA [but not efflux of poly(A)-free RNA] from isolated nuclei and the nuclear-envelope NTPase activity were strongly inhibited by Rev protein. On the other hand, transport of viral env RNA, containing the Rev-responsive element, was increased in the presence of Rev. Studying the release of RNA from closed nuclear-envelope vesicles containing entrapped RNA, the action of Rev was found to occur at the level of translocation of RNA through the nuclear pore. Evidence is presented that Rev down-regulates the NTPase-driven transport of mRNA lacking the RRE, most likely via binding to the mRNA carrier within the envelope. In contrast to the efflux of RRE-free RNA, ATP-dependent efflux of RRE-containing RNA from resealed nuclear-envelope vesicles was found to be increased, if the RNA was entrapped in the vesicles together with Rev protein. In addition, it was found that phosphorylated Rev, which is transported together with RRE-containing RNA out of the vesicles, becomes dephosphorylated during transport. In the vesicle experiments it is demonstrated for the first time that a protein selectively channels a specific mRNA across the nuclear-envelope pore complex.     

Characterisation of nuclear localisation signals of the four human core histones

The four core histones H2A, H2B, H3 and H4 are transported from the cytoplasm into the nucleus by a receptor-mediated and energy-dependent process. This nuclear transport depends on topogenic signals in the individual histone protein sequences. We have analysed such nuclear localisation signals in the core histones by means of fusion proteins consisting of individual core histones (or fragments thereof) and beta-galactosidase as a reporter protein. The results show that each of the four core histones contains several portions that are capable of mediating nuclear transport. One type of topogenic sequences consists of clustered basic amino acids in the amino terminal segments of each of the core histones. The globular portions of the core histones represent a second type of nuclear localisation signals that could only mediate nuclear transport when the whole protein domains were fused to the beta-galactosidase reporter. Fragments of the globular domains derived from each of the four core histones could not serve as nuclear localisation signals. We conclude that the nuclear targeting of core histones requires information conferred by the globular domain conformation.     

Reconstitution of nuclear protein export in isolated nuclear envelopes

Signal-dependent nuclear protein export was studied in perforated nuclei and isolated nuclear envelopes of Xenopus oocytes by optical single transporter recording. Manually isolated and purified oocyte nuclei were attached to isoporous filters and made permeable for macromolecules by perforation. Export of a recombinant protein (GG-NES) containing the nuclear export signal (NES) of the protein kinase A inhibitor through nuclear envelope patches spanning filter pores could be induced by the addition of GTP alone. Export continued against a concentration gradient, and was NES dependent and inhibited by leptomycin B and GTPgammaS, a nonhydrolyzable GTP analogue. Addition of recombinant RanBP3, a potential cofactor of CRM1-dependent export, did not promote GG-NES export at stoichiometric concentration but gradually inhibited export at higher concentrations. In isolated filter-attached nuclear envelopes, export of GG-NES was virtually abolished in the presence of GTP alone. However, a preformed export complex consisting of GG-NES, recombinant human CRM1, and RanGTP was rapidly exported. Unexpectedly, export was strongly reduced when the export complex contained RanGTPgammaS or RanG19V/Q69L-GTP, a GTPase-deficient Ran mutant. This paper shows that nuclear transport, previously studied in intact and permeabilized cells only, can be quantitatively analyzed in perforated nuclei and isolated nuclear envelopes.     

Nuclear export of tRNA

Summary Exit of tRNA from the nucleus was shown, long time ago, to be a saturable and carrier-mediated process. Nevertheless, only recently, progress in the field of nucleocytoplasmic transport gave first insight into the mechanism of tRNA nuclear export. A nuclear export receptor for tRNA (Los1p/Xpo-t), belonging to the importin (karyopherin) family, has been characterized in yeast and mammalian cells. Mature tRNA molecules can associate with Los1p/ Xpo-t and the GTP-bound form of the small GTPase Ran to form an export complex in the nucleus. This complex translocates through the nuclear-pore complexes and dissociates upon GTP hydrolysis in the cytoplasm. Genetic studies in yeast have, however, shown thatLOS1 is not essential, unless additional steps in the tRNA biogenesis pathway are impaired, suggesting the existence of additional tRNA nuclear export routes. Furthermore, modification and aminoacylation of tRNA may also be important for efficient transport of tRNA into the cytoplasm.     

Nuclear transport of histone 2b in mammalian cells is signal- and energy-dependent and different from the importin α/β-mediated process

Histone 2b nuclear transport was investigated using the digitonin-permeabilized cell system and the rat liver resealed nuclear envelope system. In permeabilized cells, maximal uptake of histone 2b is dependent on cytosolic components and an appropriate energy source. Addition of the recombinant proteins importin alpha/beta, and Ran, as well as ATP and GTP, to cytosol-depleted permeabilized cells does not enhance the uptake of histone 2b in contrast to that of nucleoplasmin serving as a control. Nuclear import of histone 2b cannot be blocked by addition of an excess of a nuclear localization signal-bearing peptide or nucleoplasmin. Similar results were obtained with resealed nuclear envelopes. As shown previously, resealed vesicles respond to the importin signal for the uptake of nuclear localization signal-bearing proteins which allows investigation of the import mechanism independent of intranuclear binding to chromatin. Uptake of histone 2b therefore seems to be an energy-requiring transport mechanism different from the import of proteins bearing a typical nuclear localization signal.     

Transport of macromolecules between the nucleus and the cytoplasm.

Nuclear transport is an energy-dependent process mediated by saturable receptors. Import and export receptors are thought to recognize and bind to nuclear localization signals or nuclear export signals, respectively, in the transported molecules. The receptor-substrate interaction can be direct or mediated by an additional adapter protein. The transport receptors dock their cargoes to the nuclear pore complexes (NPC) and facilitate their translocation through the NPC. After delivering their cargoes, the receptors are recycled to initiate additional rounds of transport. Because a transport event for a cargo molecule is unidirectional, the transport receptors engage in asymmetric cycles of translocation across the NPC. The GTPase Ran acts as a molecular switch for receptor-cargo interaction and imparts directionality to the transport process. Recently, the combined use of different in vitro and in vivo approaches has led to the characterization of novel import and export signals and to the identification of the first nuclear import and export receptors.     

Core histones and linker histones are imported into the nucleus by different pathways

Histones are the major structural proteins in eukaryotic chromosomes. This group of small very basic proteins consists of the H1 linker histones and the core histones H2A, H2B, H3 and H4. Despite their small size, the nuclear import of histones occurs by an active transport mechanism and not simply by diffusion. Histones contain several nuclear localisation signals (NLS) that can be subdivided into two different types of signal structures. We have previously shown that H1 histones are transported by a heterodimeric import receptor complex consisting of importin beta and importin 7, and we now describe the receptors required for the import of the core histones. Competition experiments using the in vitro transport assay indicate that the import pathway of the core histones differs from that of the linker histones and of nuclear proteins with classical NLS. In vitro binding assays show that each of the import receptors importin beta, importin 5, importin 7 and transportin, has the capacity to bind to any of the four core histones. Reconstitution experiments with recombinant factors indicate that each of these factors can independently serve as an import receptor for each of the core histones.