Nuclear exchange of the U1 and U2 snRNP-specific proteins

The snRNP particles include a set of common core snRNP proteins and snRNP specific proteins. In rodent cells the common core proteins are the B, D, D', E, F and G proteins in a suggested stoichiometry of B2D'2D2EFG. The additional U1- and U2-specific proteins are the 70-kD, A and C proteins and the A' and B" proteins, respectively. Previous cell fractionation and kinetic analysis demonstrated the snRNP core proteins are stored in the cytoplasm in large partially assembled snRNA-free intermediates that assemble with newly synthesized snRNAs during their transient appearance in the cytoplasm (Sauterer, R. A., R. J. Feeney, and G. W. Zieve. 1988. Exp. Cell Res. 176:344-359). This report investigates the assembly and intracellular distribution of the U1 and U2 snRNP-specific proteins. Cell enucleation and aqueous cell fractionation are used to prepare nuclear and cytoplasmic fractions and the U1- and U2-specific proteins are identified by isotopic labeling and immunoprecipitation or by immunoblotting with specific autoimmune antisera. The A, C, and A' proteins are found both assembled into mature nuclear snRNP particles and in unassembled pools in the nucleus that exchange with the assembled snRNP particles. The unassembled proteins leak from isolated nuclei prepared by detergent extraction. The unassembled A' protein sediments at 4S-6S in structures that may be multimers. The 70-kD and B" proteins are fully assembled with snRNP particles which do not leak from isolated nuclei. The kinetic studies suggest that the B" protein assembles with the U2 particle in the cytoplasm before it enters the nucleus.     

Divergence of U2 snRNA sequences in the genome of D. melanogaster.

Four different U2-snRNA genes/related sequences of D. melanogaster were cloned and characterized. The sequences of all four genes suggest that they were generated by a DNA-mediated mechanism. These genes/related sequences were found to be located in two loci, each locus containing two U2 snRNA sequences. Using coding sequences as well as flanking sequences as hybridization probes against polytene chromosomes of D. melanogaster Oregon R we were able to map these loci separately at positions 34BC and 84C. By Northern analysis we observed that the quantities of U2- and U1-snRNA are coordinated and change during the embryonic development of the fly.     

Crystal structures of two Sm protein complexes and their implications for the assembly of the spliceosomal snRNPs

The U1, U2, U4/U6, and U5 small nuclear ribonucleoprotein particles (snRNPs) involved in pre-mRNA splicing contain seven Sm proteins (B/B', D1, D2, D3, E, F, and G) in common, which assemble around the Sm site present in four of the major spliceosomal small nuclear RNAs (snRNAs). These proteins share a common sequence motif in two segments, Sm1 and Sm2, separated by a short variable linker. Crystal structures of two Sm protein complexes, D3B and D1D2, show that these proteins have a common fold containing an N-terminal helix followed by a strongly bent five-stranded antiparallel beta sheet, and the D1D2 and D3B dimers superpose closely in their core regions, including the dimer interfaces. The crystal structures suggest that the seven Sm proteins could form a closed ring and the snRNAs may be bound in the positively charged central hole.     

The snRNP core assembly pathway: identification of stable core protein heteromeric complexes and an snRNP subcore particle in vitro.

Stable association of the eight common Sm proteins with U1, U2, U4 or U5 snRNA to produce a spliceosomal snRNP core structure is required for snRNP biogenesis, including cap hypermethylation and nuclear transport. Here, the assembly of snRNP core particles was investigated in vitro using both native HeLa and in vitro generated Sm proteins. Several RNA-free, heteromeric protein complexes were identified, including E.F.G, B/B'.D3 and D1.D2.E.F.G. While the E.F.G complex alone did not stably bind to U1 snRNA, these proteins together with D1 and D2 were necessary and sufficient to form a stable U1 snRNP subcore particle. The subcore could be chased into a core particle by the subsequent addition of the B/B'.D3 protein complex even in the presence of free competitor U1 snRNA. Trimethylation of U1 snRNA's 5' cap, while not observed for the subcore, occurred in the stepwise-assembled U1 snRNP core particle, providing evidence for the involvement of the B/B' and D3 proteins in the hypermethylation reaction. Taken together, these results suggest that the various protein heterooligomers, as well as the snRNP subcore particle, are functional intermediates in the snRNP core assembly pathway.     

Chromosomal assignment of genes controlling salt-soluble proteins (albumins and globulins) in wheat and related species

Summary Salt-soluble proteins from the endosperms of wheat, barley, and rye have been separated by nonequilibrium electrofocusing x electrophoresis. Genes encoding 14 of the 25 components observed in wheat have been unambiguously assigned to 10 different chromosomes (1B, 3B, 3D, 4A, 4D, 5B, 6B, 6D, 7B, 7D) by analysis of the compensated nulli-tetrasomic series. Five more wheat proteins seem to be controlled by group 2 chromosomes. Analysis of wheat-barley and wheat-rye addition lines has led to the location of genes for 6 out of 20 barley proteins in 4 different chromosomes (1H, 3H, 4H, 6H; 1H is homoeologous to group 7 chromosomes of wheat) and of genes for 5 out of 20 rye proteins in two different chromosomes (2R, 4R). The relationship between the proteins reported here and previously characterized ones is discussed.     

Chromosome mapping of Rmp-4, a gonad-dependent gene encoding host resistance to mousepox.

DBA/2 (D2) mice are susceptible and C57BL/6 (B6) mice are resistant to lethal mousepox. A congenic resistant strain, D2.B6-Rmp-4r (D2.R4), was developed by serially backcrossing male mice that survived ectromelia virus infection with D2 mice, beginning with (B6 x D2)F1 mice. Male D2.R4 mice were at least 300-fold more resistant to lethal mousepox than male D2 mice. Female D2.R4 mice were 100-fold more resistant than male D2.R4 mice and 500-fold more resistant than female D2 mice. Neonatal gonadectomy prevented development of resistance in D2.R4 mice of both sexes. Differences in resistance between strains and between sexes correlated with restriction of virus replication in spleen and liver, but gender differences were less evident in liver than in spleen. High-resolution interval mapping of the 19 autosomes of D2.R4 mice using dispersed informative microsatellites as marker loci revealed a segment of distal chromosome 1 to be of B6 origin. Haplotypes for a marker locus, D1Mit57, from the differential segment were determined in (D2.R4 x D2)F1 x D2 backcross mice, which were then infected with ectromelia virus. Significantly more heterozygotes than homozygotes survived ectromelia virus infection in both sexes. Whereas nearly all surviving males were heterozygotes, 44% of surviving females were homozygotes. These results indicate that resistance in D2.R4 mice is determined by a gonad-dependent gene on distal chromosome 1, provisionally named Rmp-4, and by an ovary-dependent factor that is not genetically linked to Rmp-4.     

Recombinant haplotype H-2 in mice of the congenic resistant strain B10.D1(R108)/Y

Recombinant H-2 haplotype of mouse strain B10.D1(R108)/Y (symbol R108) obtained in experiments with skin grafting in the course of developing the CR B10.D1/Y strain (strain DBA/LacY--the donor of H-2q) was studied. Strains with recombinant H-2 haplotypes a, h2, g1, i3, i5, i7, m, y1 were used. Alleles of different H-2 (K, I, D) regions were determined according to the presence or absence of genetic complementation in the F1 test with skin grafts. R108 recombinant was studied by serological methods with panel of anti-H-2 sera. Anti-H-2Kb (H-2.33) and anti-H-2Dq (H-2.30) monospecific antisera were used in microcytotoxicity test and in absorption experiments in vitro. It was concluded that crossing over between H-2b and H-2q chromosomes, which led to formation of recombinant H-2 haplotype of R108 mice, occurred at I region, between IA and IC subregions. The H-2 complex of R108 line has KbIAbIJ?IE?ICqSqDq alleles. bq1 symbol was proposed for the H-2 haplotype of B10.D1(R108)/Y strain.     

Increase of MnSOD expression and decrease of JNK activity determine the TNF sensitivity in bcl2-transfected L929 cells.

To investigate the protection mechanism of Bcl-2 against tumour necrosis factor (TNF)-mediated cell death, the bcl2 gene was transfected into the L929 cells and stably expressed. Two clones having different sensitivity among bcl2-transfected L929 clones had been isolated, and termed clone R1 and R2. It was observed that activation of manganese superoxide dismutase (MnSOD) and suppression of Jun kinase of clone R1 and R2 were correlated with protection from TNF cytotoxicity. Upon treatment with TNF, clone R1 and R2 were more resistant than control L929 cells against TNF cytotoxicity and the protective effect of clone R1 was stronger than clone R2. However, in case of TNF plus actinomycin D treatment, clone R1 was still resistant against TNF cytotoxicity, whereas clone R2 became more sensitive than control L929 cells. The JNK activities of clone R1 and R2 were suppressed upon TNF treatment and in case of TNF plus actinomycin D treatment, clone R2 showed a marked increase in JNK activities and had higher activity than control L929 cells. The specific activities of MnSOD of clone R1 and R2 upon TNF treatment were 70 U/ml and 33 U/ml, respectively, while the MnSOD activity was not detectable in control L929 cells. When TNF and actinomycin D were treated simultaneously, MnSOD activity was not detectable in control L929 cells and bcl2 -transfected L929 cells (clone R1, R2). Consistent with these results, both clone R1 and R2 showed higher levels of MnSOD mRNA expression than control L929 cells after TNF treatment. These data suggest that suppression of Jun kinase and increase of MnSOD may be involved in inhibitory action of Bcl-2 against TNF, and the balance between MnSOD and JNK signalling pathway may be an important factor for the protection of bcl2-transfected L929 cells from TNF cytotoxicity.     

Hetero-oligomerization between GABAA and GABAB receptors regulates GABAB receptor trafficking

The neurotransmitter gamma-aminobutyric acid (GABA) mediates inhibitory signaling in the brain via stimulation of both GABA(A) receptors (GABA(A)R), which are chloride-permeant ion channels, and GABA(B) receptors (GABA(B)R), which signal through coupling to G proteins. Here we report physical interactions between these two different classes of GABA receptor. Association of the GABA(B) receptor 1 (GABA(B)R1) with the GABA(A) receptor gamma2S subunit robustly promotes cell surface expression of GABA(B)R1 in the absence of GABA(B)R2, a closely related GABA(B) receptor that is usually required for efficient trafficking of GABA(B)R1 to the cell surface. The GABA(B)R1/gamma2S complex is not detectably functional when expressed alone, as assessed in both ERK activation assays and physiological analyses in oocytes. However, the gamma2S subunit associates not only with GABA(B)R1 alone but also with the functional GABA(B)R1/GABA(B)R2 heterodimer to markedly enhance GABA(B) receptor internalization in response to agonist stimulation. These findings reveal that the GABA(B)R1/gamma2S interaction results in the regulation of multiple aspects of GABA(B) receptor trafficking, allowing for cross-talk between these two distinct classes of GABA receptor.     

Subunit-specific modulation of [(3)H]MK-801 binding to NMDA receptors mediated by dopamine receptor ligands in rodent brain

Dopamine D1 receptor (D1R) ligands may directly interact with the NMDA receptor (NMDAR), but detailed knowledge about this effect is lacking. Here we identify D1R ligands that directly modulate NMDARs and examine the contributions of NR2A and NR2B subunits to these interactions. Binding of the open channel blocker [(3)H]MK-801 in membrane preparations from rat- and mouse brain was used as a biochemical measure of the functional state of the NMDAR channel. We show that both D1R agonist A-68930 and dopamine receptor D2 antagonist haloperidol can decrease [(3)H]MK-801 binding with increased potency in membranes from the NR2A(-/-) mice (i.e. in membranes containing NR2B only), as compared to the inhibition obtained in wild-type membranes. Further, a wide range of D1R agonists such as A-68930, SKF-83959, SKF-83822, SKF-38393 and dihydrexidine were able to decrease [(3)H]MK-801 binding, all showing half maximal inhibitory concentrations ~20 μM, and with significant effects occurring at or above 1 μM. With membranes from D1R(-/-) mice, we demonstrate that these effects occurred through a D1R-independent mechanism. Our results demonstrate that dopamine receptor ligands can selectively influence NR2B containing NMDARs, and we characterize direct inhibitory NMDAR effects by different D1R ligands.     

Kinetic studies on the cleavage of oligouridylic acids and poly U by bovine pancreatic ribonuclease A

Kinetic parameters, Km and Vmax for the transesterification of oligouridylic acid, (Up)nU greater than p (n=0-4), by RNase A were measured spectrophotometrically at pH 7.0 and 25 degrees C. The kinetic parameters, pKm and log Vmax increased with increase in the chain length (n), and seemed to be almost constant with substrates having n greater than or equal to 2. The contribution of each subsite to the binding was estimated according to Hiromi's theory. The subsite affinities for (B1, R1, P1)+(B2, R2, P2) and (B3, R3, P3) are 8.03 kcal and 0.72 kcal/mol, respectively, and those for (B4, R4, P4) and (B5, R5, P5) are less than 0.5 kcal/mol. Therefore, we postulate that the size of the RNase A active site is about 3 nucleotides in length. Transesterification of poly U by RNase A was followed spectrophotometrically. The reaction is markedly influenced by ionic strength. At lower ionic strength, the v0-S curve of poly U cleavage was sigmoidal and cooperative, and it became less cooperative at higher ionic strength. Since the estimated Vmax value for poly U cleavage at ionic strength of 0.1 was more than 20 times larger than that of oligouridylic acids cleavage, we propose a non-specific interaction of poly U anion with cationic groups on the surface of the enzyme, modulating the conformation of active site, and thus increasing the activity at low ionic strength. The interaction decreases at higher ionic strength due to the interaction of counter anions with the non-specific sites.     

Molecular cytogenetic analyses of hexaploid lines spontaneously appearing in octoploid Triticale

Genome characterization of 14 hexaploid lines that spontaneously appeared in octoploid Triticales was carried out by sequential genomic in situ hybridization and fluorescence in situ hybridization, high molecular weight glutenin subunits and SSR marker analyses. All of the lines showed a chromosome constitution of complete A and B genomes, and a composite genome consisting of the chromosomes of D and R genomes. The composite genome of the 11 lines consisted of chromosomes 1R, 2D, 3R, 4R, 5R, 6R and 7R, that of the two lines were 1D, 2D, 3R, 4R, 5R, 6R and 7R, and that of one line was 1R, 2D, 3R, 4R, 5R, 6D and 7R. The incompatibility of the D and R genomes in common wheat genetic background, preferential retention of chromosome 2D and importance of these lines for the development of hexaploid Triticale are discussed in this report.     

Physical mapping and molecular-cytogenetic analysis of substitutions and translocations involving chromosome 1D in synthetic hexaploid triticale

 Chromosome 1D, which carries the advantageous alleles of glutenin and gliadin, attracts major interest with respect to improving the bread-making quality of triticale. Eighty-one BC₁F₄ lines from different primary and secondary hexaploid triticale crosses were selected for 1D chromatin analysis using SDS-PAGE and C-banding. In situ hybridization and RFLP-based comparative physical mapping of group 1 chromosomes revealed 20 lines with complete 1D (1A) substitutions. Nine 1D (1B) substitutions, six 1D (1R) substitutions and one 1D addition line were also selected. Three lines were pure AABBRR hexaploids without any D-genome chromosomes. For the remaining 42 lines (51.8%), a wide spectrum of 20 different recombinations between chromosomes 1A and 1D was uncovered. Altogether, they were generated without any earlier irradiation, tissue culture or genetic induction of chromosome pairing. In addition, 14 translocations between 1B/1D, 1A/1R, 1B/1R, 1D/1R and 1A/1B were detected. Considerable variability for sedimentation values was found, with the highest sedimentation values among lines with complete 1D chromosomes. The implications of using triticale as a model for generating compensating chromosome rearrangements in defined homoeologous groups and the breeding potential of D-genome chromatin introgressed into triticale with improved sedimentation values are discussed. Received: 27 July 1998 / Accepted: 5 August 1998     

Targeting DYRK1A/B kinases to modulate p21-cyclin D1-p27 signalling and induce anti-tumour activity in a model of human glioblastoma

The dual-specificity tyrosine-regulated kinases DYRK1A and DYRK1B play a key role in controlling the quiescence-proliferation switch in cancer cells. Serum reduction of U87MG 2D cultures or multi-cellular tumour spheroids induced a quiescent like state characterized by increased DYRK1B and p27, and decreased pRb and cyclin D1. VER-239353 is a potent, selective inhibitor of the DYRK1A and DYRK1B kinases identified through fragment and structure-guided drug discovery. Inhibition of DYRK1A/B by VER-239353 in quiescent U87MG cells increased pRb, DYRK1B and cyclin D1 but also increased the cell cycle inhibitors p21 and p27. This resulted in exit from G0 but subsequent arrest in G1. DYRK1A/B inhibition reduced the proliferation of U87MG cells in 2D and 3D culture with greater effects observed under reduced serum conditions. Paradoxically, the induced re-expression of cell cycle proteins by DYRK1A/B inhibition further inhibited cell proliferation. Cell growth arrest induced in quiescent cells by DYRK1A/B inhibition was reversible through the addition of growth-promoting factors. DYRK inhibition-induced DNA damage and synergized with a CHK1 inhibitor in the U87MG spheroids. In vivo, DYRK1A/B inhibition-induced tumour stasis in a U87MG tumour xenograft model. These results suggest that further evaluation of VER-239353 as a treatment for glioblastoma is therefore warranted.     

Does histidine 332 of the D1 polypeptide ligate the manganese cluster in photosystem II? An electron spin echo envelope modulation study

The tetranuclear manganese cluster in photosystem II is ligated by one or more histidine residues, as shown by an electron spin echo envelope modulation (ESEEM) study conducted with [(15)N]histidine-labeled photosystem II particles isolated from the cyanobacterium Synechocystis sp. strain PCC 6803 [Tang, X.-S., Diner, B. A., Larsen, B. S., Gilchrist, M. L., Jr., Lorigan, G. A., and Britt, R. D. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 704-708]. One of these residues may be His332 of the D1 polypeptide. Photosystem II particles isolated from the Synechocystis mutant D1-H332E exhibit an altered S(2) state multiline EPR signal that has more hyperfine lines and narrower splittings than the corresponding signal in wild-type PSII particles [Debus, R. J., Campbell, K. A., Peloquin, J. M., Pham, D. P., and Britt, R. D. (2000) Biochemistry 39, 470-478]. These D1-H332E PSII particles are also unable to advance beyond an altered S(2)Y(Z)(*) state, and the quantum yield for forming the S(2) state is very low, corresponding to an 8000-fold slowing of the rate of Mn oxidation by Y(Z)(*). These observations are consistent with His332 being close to the Mn cluster and modulating the redox properties of both the Mn cluster and tyrosine Y(Z). To determine if D1-His332 ligates the Mn cluster, we have conducted an ESEEM study of D1-H332E PSII particles. The histidyl nitrogen modulation observed near 5 MHz in ESEEM spectra of the S(2) state multiline EPR signal of wild-type PSII particles is substantially diminished in D1-H332E PSII particles. This result is consistent with ligation of the Mn cluster by D1-His332. However, alternate explanations are possible. These are presented and discussed.     

Monospecific antibodies reveal details of U2 snRNP structure and interaction between U1 and U2 snRNPs.

Monospecific antibodies directed against several U small nuclear ribonucleoprotein (U snRNP) particle proteins were affinity purified from a patient's anti-(U1,U2)RNP serum. These were used to demonstrate that: (i) proteins equivalent to the mammalian U2 snRNP-specific A' and B" proteins are present in Xenopus laevis oocytes; (ii) both proteins A' and B" have the same structural requirements for binding to U2 snRNA; (iii) proteins B, B' and D have the same structural requirement for binding to U2 snRNA; (iv) using very high specific activity RNA probes it is possible to detect a fraction of either U1 or U2 snRNA precipitable by antibodies directed against proteins specific for the other U snRNP, indicating an interaction between U1 and U2 snRNPs. The structural requirements of this interaction were studied for the U2 snRNP. All changes made to U2 snRNA or snRNP structure resulted in loss of the interaction with U1 snRNP.     

Statice Virus Y - a Virus related to Bean Yellow Mosaic and Clover Yellow Vein Viruses

The authors are greatly indebted to the Deutsche Forschungsgemeinschaft for financial support, to Miss B. M urthlm , Mrs. U. H erzberg , Mrs. B. H ane and Miss P. R ahse for reliable technical assistance and to Dr. M. H ollings , Dr. R. O. H ampton and Mr. D. Z. M aat for gifts of antisera.