Nuclear Cap Research Articles

A yeast cap binding protein complex (yCBC) acts at an early step in pre-mRNA splicing.

The function in splicing of a heterodimeric nuclear cap binding complex (yCBC) from the yeast Saccharomyces cerevisiae has been examined. Immunodepletion of splicing extracts with antibodies directed against one component of the complex, yCBP80, results in the efficient co-depletion of the second component, yCBP20, producing CBC-deficient splicing extract. This extract exhibits strongly reduced splicing efficiency and similar reductions in the assembly of both spliceosomes and of the earliest defined precursors to spliceosomes, commitment complexes. The addition of highly purified yCBC substantially restores these defects. These results, together with other data, suggest that CBCs play a highly conserved role in the recognition of pre-mRNA substrates at an early step in the splicing process.

Nuclear Cap Binding Protein Maps Close to the Xeroderma Pigmentosum Complementation Group A (XPA) Locus in Human and Mouse

This report describes the localization of the nuclear cap binding protein (NCBP) to human chromosome 9 and mouse chromosome 4 using somatic cell hybridization analysis. NCBP plays an important role in the splicing and transport of messenger-RNA. 11 refs., 1 fig.

Identification of the factors that interact with NCBP, an 80 kDa nuclear cap binding protein.

It has been shown that the monomethylated cap structure plays important roles in pre-mRNA splicing and nuclear export of RNA. As a candidate for the factor involved in these nuclear events we have previously purified an 80 kDa nuclear cap binding protein (NCBP) from a HeLa cell nuclear extract and isolated its full-length cDNA. In this report, in order to obtain a clue to the cellular functions of NCBP, we attempted to identify a factor(s) that interacts with NCBP. Using the yeast two-hybrid system we isolated three clones from a HeLa cell cDNA library. We designated the proteins encoded by these clones NIPs (NCBP interacting proteins). NIP1 and NIP2 have an RNP consensus-type RNA binding domain, whereas NIP3 contains a unique domain of Arg-Glu or Lys-Glu dipeptide repeats. We also show that NCBP requires NIP1 for binding to the cap structure. Possible roles of NIPs in cap-dependent nuclear processes are discussed.

A nuclear cap binding protein complex involved in pre-mRNA splicing

A cap-binding protein complex (CBC) present in the nuclei of HeLa cells has been characterized. Purified CBC consists of two previously identified proteins, CBP80 and CBP20. These proteins are shown to cofractionate to apparent homogeneity and to be coimmunoprecipitable with anti-CBP80 antibodies. Analysis of the inhibition of pre-mRNA splicing in vitro and in vivo by chemically modified analogs of the cap structure, and of the binding of these analogs to CBC in vitro, suggests a role for the complex in splicing. Extracts immunodepleted of CBC do not efficiently splice an adenoviral pre-mRNA owing to blockage of an early step in splicing complex formation. CBC may therefore play a role in pre-mRNA recognition.

Cloning of a complementary DNA encoding an 80 kilodalton nuclear cap binding protein.

It has been shown that the monomethylated cap structure plays important roles in nuclear events. The cap structure has been implicated in the enhancement of pre-mRNA splicing. More recently, this structure has also been suggested to facilitate RNA transport from the nucleus to the cytoplasm. We have previously identified and purified an 80kD Nuclear Cap Binding Protein (NCBP) from a HeLa cell nuclear extract, which could possibly mediate these nuclear activities. In this report, we describe cloning of complementary DNA (cDNA) encoding NCBP. The partial protein sequences of NCBP were determined, and the full-length cDNA of NCBP was isolated from HeLa cDNA libraries. This cDNA encoded an open reading frame of 790 amino acids with a calculated molecular mass of 91,734 daltons, which contained most of the determined protein sequences. However, the protein sequence had no significant homology to any known proteins. Transfection experiments demonstrated that the epitope-tagged NCBP, transiently expressed in HeLa cells, was localized exclusively in the nucleoplasm. Similar experiments using a truncated NCBP cDNA indicated that this nuclear localization activity is conferred by the N-terminal 70 amino-acid region.

The incidence of nuclear caps in dyschromia

The incidence of nuclear caps in dyschromia

Quantitative age-related changes on nuclear invaginations of neocerebellar Purkinje cells.

The aim of this study was to provide stereological data for seeking whether Purkinje neurones increase their nuclear envelope invaginations with age. The nuclear parameters considered were: ratio of absolute surface area to volume, surface density and absolute surface area. The first two parameters did not significantly change, but the absolute surface area exhibited marked variations. Regression studies showed no significant linear correlations between any of the parameters and age; so, the increase in number and/or in depth of the invaginations was not suggested. The association of invaginations with a nuclear cap of Nissl substance is a constant feature, stressing that the physiological importance of the complex is maintained throughout the ageing process.

Histochemical analysis of rat testicular glycoconjugates. 1. Subsets of N-linked saccharides in seminiferous tubules.

The distribution of N-linked glycans in rat testis has been probed using a panel of lectins derived from Galanthus nivalis (snowdrop, GNA), Canavalia ensiformis (jack bean, Con A), Lens culinaris (lentil, LCA), Pisum sativum (garden pea, PSA) and Phaseolus vulgaris, erythro- and leucoagglutinins (kidney bean, ePHA and lPHA). Several classes of N-linked glycan were identified in the spermatogenic series, and during differentiation into spermatozoa they altered in both their pattern of distribution and relative abundance. A population of tetra-antennary, non-bisected, complex glycans, detected by lPHA, was lost during the transition from spermatogonia to spermatocytes, while high-mannose structures were acquired; these were most abundant in spermatocytes, as were bi- and tri-antennary complex, non-bisected glycans, the latter becoming increasingly abundant on acrosomes and spermatozoa. Their bisected counterparts were more generally expressed throughout spermatogenic cells, although marked localization onto acrosomes and nuclear caps was again seen. Transition from spermatocytes to spermatids involved mainly changes of the acrosomal granule and nuclear cap, which were carried through to the final stages of differentiation. Sertoli cell surfaces and cytoplasmic granules showed a high level of N-glycan expression.

Histochemical analysis of rat testicular glycoconjugates. 2. Beta-galactosyl residues in O- and N-linked glycans in seminiferous tubules.

Rat testes have been examined with a panel of lectins that bind specifically to oligosaccharide sequences having terminal or subterminal beta-galactosyl residues in O-linked glycans, or in the outer chains of complex N-linked glycans: Arachis hypogaea (peanut, AHA), Erythrina cristagalli (coral tree, ECA), Ricinus communis (castor bean, RCA120) and Abrus precatorius (jequirity bean, APA) agglutinins. Pretreatment of sections with neuraminidase, beta-galactosidase and removal of alkali-labile O-linked sequences by beta-elimination allowed the structure of these glycans to be further explored. In spermatogonia and spermatocytes there was little evidence of glycans terminating in beta-galactosyl residues, although these were present at non-reducing terminals as sialylgalactosides. The acrosome contained two subsets of O-linked glycans terminating in sialylgalactosides, while the nuclear cap showed at least two subsets of N-linked sialylgalactosyl as well as O-linked glycans. Spermatozoa exhibited minor changes in the pattern of glycosylation, although the overall pattern of beta-galactosyl expression was similar. Binding to Sertoli cells showed the presence of some unsubstituted beta-galactosyl terminals on O-linked glycans but few such N-linked residues, while terminal beta-galactosides were scanty in tubular basement membranes.

Redistribution of nuclear lamin A is an early event associated with differentiation of human promyelocytic leukemia HL-60 cells.

The nuclear lamina of mammalian somatic cells is characterized by the constitutive presence of lamin B polypeptides while the appearance of lamins A and C generally occur during establishment of a differentiated phenotype. We have used antibodies specific to the unique carboxy-terminal domain of lamin A, i.e. distinct from the shared domains of lamins A and C, to study the individual behaviour of lamin A during establishment of a macrophage phenotype in human HL-60 cells. Lamin A was present as a nuclear cap in the majority of undifferentiated cells and it was redistributed to a full peripheral nuclear location very early after induction of differentiation by phorbol esters, even in the presence of a protein synthesis inhibitor. Induction of the cells into a reversible precommitment state by bromodeoxyuridine was accompanied by a similar redistribution of lamin A that however reverted to a cap after removal of inducer. No changes were observed in the uniform peripheral nuclear location of lamin C under all of these conditions. These results strongly suggest that lamin A plays a role in the early events of cell differentiation. Taken together with previous results on the interaction of A-type lamins with chromatin, these findings offer experimental evidence consistent with the proposed role of A-type lamins, and particularly lamin A, in the process of chromatin reorganization that accompanies the expression of a differentiated phenotype.

Ultrastructure of spermiogenesis of Saudian reptiles. 6. The sperm head differentiation in Agama adramitana.

In addition to the common features of spermiogenesis in Reptilia, new structures are seen in Agama adramitana that have not been described in previous studies. Two acrosomal granules are observed in the acrosomal vesicle. The posterior acrosomal granule differentiates as an anterior electron-dense material in the acrosome, and the anterior one forms a posterior translucent component of the acrosome. Also, the subacrosomal granule spreads in the subacrosomal space as a subacrosomal nuclear cap.

Synthesis of γ-[4-(benzoylphenyl)methylamido]-7-methylguanosine 5′-triphosphate, a photoaffinity-label for cap-binding proteins

An easy and rapid method for the synthesis of the photoreactive cap analogue γ-[4-(benzoylphenyl)methylamido]-7-methylguanosine-5′-triphosphate (BP-m 7GTP) has been developed. Synthesis of [γ- 32P]BP-m 7GTP was carried out as a two-step procedure. The first step included the phosphorylation of GDP with [ 32P]orthophosphate to [γ- 32P]GTP and its methylation to [γ- 32P]m 7GTP. After purification, [γ- 32P]m 7GTP was condensed with 4-aminomethyl-benzophenone-HCl to [γ- 32P]BP-m 7GTP. Using this probe, specific labeling of nuclear cap binding proteins is demonstrated.

The fine structure of the zoospore of Sorochytrium milnesiophthora

As a part of an investigation of Sorochytrium milnesiophthora Dewel, a parasite of the tardigrade Milnesium tardigradum, the ultrastructure of the zoospore was examined. In overall organization, the zoospore is similar to the spores of the Blastocladiales. The nucleus is conical and located in the posterior third of the cell. A large nuclear cap is anterior to the nucleus. In an arrangement unique for members of the Blastocladiales, the nuclear cap contains, in addition to aggregated ribosomes, a single complex mitochondrion, lipid globules, and microbodies. Portions of the nuclear cap extend posteriorly and partially surround the kinetosome. The kinetosome has nine anterior rootletlike projections that link it to the mitochondrion and nine posterior kinetosomal props that attach it to the plasmalemma. It lies at the base of the nucleus as an anchor for a single, posterior whiplash flagellum. A nonkinetosomal centriole is positioned at an approximate 45° angle to the kinetosome. In the peripheral cytoplasm, there are three structurally distinct types of inclusions hypothesized to be adhesion vesicles, phosphate storage bodies, and gamma-like particles. Therefore, the ultrastructure of the zoospore supports the inclusion of this fungus in the Blastocladiales. Key words: Blastocladiales, zoospore, ultrastructure.

A nuclear cap binding protein from HeLa cells

We have identified a cap binding protein in a HeLa nuclear extract using a gel mobility shift assay probed with capped RNA. Subcellular fractionation of HeLa cells revealed that the majority (about 70%) of the cap binding activity is present in the nuclear extract, about 20% is in the cytoplasmic S100 fraction, and almost none in the ribosome-high salt wash fraction, indicating that the protein in active form localizes mainly in the nuclei. Competition experiments with various cap analogues showed that the G(5')ppp(5')N-blocking structure as well as the methyl residue at the N7 position of the blocking guanosine is important for the binding of this protein, and that the trimethylguanosine cap structure which exists at the 5' termini of many snRNAs is not recognized by this protein. Immunoprecipitation experiments using various anti-snRNP antibodies suggested that this protein is partially associated with U2 snRNP. We purified this protein to near homogeneity from a HeLa nuclear extract by several chromatographic procedures including capped RNA-Sepharose chromatography. The purified protein shows molecular weight of 80 kilodaltons, as judged by SDS gel electrophoresis, and binds specifically to the cap structure.

The axon reaction of lamprey spinal interneurons

Axotomy and partial denervation of giant interneurons (GIs) and lateral cells (LCs) were produced by complete spinal transection in the larval lamprey spinal cord. Both cell types demonstrated a reduction in cytoplasmic basophilia, increase in cell size, nuclear eccentricity, and formation of a chromophilic nuclear cap. This was quantified in the case of cell diameter. During the first 8 weeks of recovery, the GIs with the largest diameters were found progressively further from the scar and this peak change moved at approximately 0.5 mm/day. The increase in size of GIs remained up to 20 weeks post-transection, long after the time required for their axons to regenerate across the scar and form functioning synapses. GIs injected intracellularly with horseradish peroxidase (HRP) also showed this increase in diameter as well as a simplification of their dendritic trees. Intracellular recordings from GIs revealed changes in the frequency and amplitude of spontaneous synaptic input. In the first two weeks after transection, spontaneous excitatory postsynaptic potentials (EPSPs) and inhibitory postsynaptic potentials (IPSPs) were less frequent than in control cells. After 6 weeks of recovery they became more frequent than in control cells. EPSPs predominated in axotomized GIs, while in control cells they constituted only 36% of the total of spontaneous potentials. A reversible increase in the amplitude of these EPSPs occurred at 3–4 weeks of recovery time. The resting membrane potential was significantly reduced by the 6th week after transection and returned to normal after the 22nd week.

The synchronization and timing of cytological events during gametogenesis in Allomyces macrogynus

A method is described with which the cytological events during gamete formation in Allomyces macrogynus can be examined in detail. Synchronous development was achieved using a microdroplet slide culture system to produce gametangia that were all of the same developmental age. Game-tothallic cultures were produced by inoculating 100 meiospores/100 μl of medium onto microscope slides and incubating the microdroplet cultures at 22°C in moist chambers. Forty-eight hours after inoculation, small hyphal fans were observed with each terminal hypha containing male and female gametangial pairs. These cultures showed a much more synchronous release of gametes than did other culture methods tried (i.e., liquid suspension cultures and agar plate cultures). After induction of gametogenesis in samples derived from microdroplet cultures, the time courses for lipid crown stage breakdown, flagellum formation, cytoplasmic cleavage, and nuclear cap formation were determined using transmission electron microscopy. This information was used to construct a timetable for these specific cytological events during gamete formation. The higher degree of synchrony of gametogenesis achieved with this method allows specific temporal events to be examined with much greater precision than has been previously possible.

Identification of nuclear cap specific proteins in HeLa cells.

Two polypeptides of apparent molecular mass of 20 and 115 kilodaltons in nuclear fractions from HeLa cells were shown to recognize and be crosslinked to the cap structure of eukaryotic mRNAs in a cap-dependent fashion. Crosslinking of the 20 and 115 kDa polypeptides was sensitive to inhibition by low concentrations of the cap analogue m7GDP and resistant to inhibition by high KCl concentrations. In addition, crosslinking of these polypeptides to the cap structure occurred in nuclear extracts prepared from poliovirus-infected cells, under conditions where cytoplasmic cap binding proteins were incapable of interacting with the mRNA cap structure. The possible function of nuclear cap binding proteins is discussed.

Effect of (D-Trp 6,Pro 9-NEt)-GnRH on the function and ultrastructure of pituitary and testis in male rats

Groups of ten adult male Wistar rats were treated chronically with GnRH agonist for different durations. The responses of pituitary and testis to GnRH agonist were evaluated by serum and testicular testosterone, serum and pituitary LH, and testicular LH receptors.Testicular testosterone and LH receptors were decreased very significantly under our experimental regimen, irrespective of the duration of the treatment Serum testosterone levels were reduced after 10 and 15 injections, but only rats receiving 10 injections showed a significant increase in serum LH levels and decrease of pituitary LH content. The type II gonadotropes of the pars distalis of pituitary were increased in number but showed a higher degree of dilated smooth endoplasmic reticulum with few secretory granules. Many large lipid droplets appeared in the cytoplasms of the Leydig cells. Degenerative changes were observed in most seminiferous tubules following GnRH agonist treatment. Obvious abnormal acrosome and nuclear caps of some spermatids were also observed. The functional changes caused by GnRH agonist coincided with their ultrastructural appearance.

Ectopic Purkinje cells in the cerebellar white matter of normal adult rodents: a Golgi study.

In Golgi/Río-Hortega preparations of rat and rabbit cerebellar vermis we have occasionally found isolated ectopic Purkinje cells in the white matter. They were located beneath the bases of the folia and their dendritic branches extended within the confines of the white matter without penetrating into the overlying cortical layers. The general morphology of these ectopic cells was variable, particularly in the extension and shape of the dendritic trees, but all of them exhibited a lower density of dendritic branches than normal Purkinje cells. The less-developed ectopic neurons had multipolar dendritic trees with nonplanar branches irregularly studded with spines. The well-developed ones displayed a more extensive arborization of their processes and they usually preserved some morphological features of normal cortical Purkinje cells: distal dendritic branches studded with numerous spines, a pear-shaped soma, clearly defined morphological polarity and a tendency to display planar arrangement of the dendritic arbors. In semithin sections these neurons also showed cytological features of normal Purkinje cells, such as the Nissl substance forming a nuclear cap oriented toward the dendritic pole. We suggest that the abnormal location of the neurons results from a disorder of Purkinje cell migration which occurs naturally during the prenatal development of the cerebellum. The possible morphogenetic mechanisms involved in the migration and differentiation of these ectopic neurons are also discussed.

A new chytridiomycete parasitizing the tardigrade Milnesium tardigradum

The life cycle of a chytridiomycete, Sorochytrium milnesiophthora gen. et sp. nov., infecting the tardigrade Milnesium tardigradum, is described. The zoospores are posteriorly uniflagellate and ovoid, and possess a central mass similar to a nuclear cap. To initiate an endobiotic infection they attach to the cuticle of the host, encyst, and generate an appressorium. The appressorium forms a penetration tube which crosses the cuticle to the epidermis and enlarges at the tip into a spherical thallus. Concomitantly, vacuoles replace the cytoplasm of the cyst and appressorium. As the thallus enlarges, it moves into the body cavity and cleaves into segments. The segments separate and round up into incipient sporangia. The incipient sporangia develop branching rhizoids in conjunction with host death. After a period of growth the sporangia form inoperculate exit papillae which penetrate the host cuticle. Zoospores exit individually and fully formed. The fungus can develop a polycentric phase when freshly collected, dead hosts containing sporangia are cultured in habitat water or on nutrient agar. The growth is extramatrical, covering the surface of the old host. It is rhizoidal, branching, and nonseptate with numerous intercalate incipient sporangia bearing rhizoids. On nutrient media the thallus grows indefinitely while in habitat water the incipient sporangia mature and discharge motile spores. The spores frequently have two to five flagella and are larger than those of the endobiotic colonial phase. A similar polycentric growth develops when motile spores are isolated on nutrient medium and suggests that the extramatrical growth on the host originates from encysted spores.