Human RNaseP RNA and nucleolar 7-2 RNA share conserved ‘To’ antigen-binding domains

RNase P in both prokaryotes and eukaryotes is a ribonucleoprotein that cleaves tRNA precursors to generate the 5 termini of the mature tRNAs. Many patients with autoimmune diseases produce antibodies against a 40 kDa protein (designatedTo orTh antigen) which is an integral component of eukaryotic RNaseP as well as nucleolar 7-2 RNP which is identical to the mitochondrial RNA processing (MRP) RNP. Interestingly, theTo antigen found in human cells and the C5 protein, the only protein component ofE. coli RNaseP, are antigenically related. In this study, we show that a 56 nucleotide-long sequence, corresponding to nucleotides 20–75 near the 5 end of human RNaseP RNA, is sufficient to bind theTo antigen. We previously showed that the humanTo antigen binds to a short distinct structural domain near the 5 end of human 7-2/MRP RNA. There is no obvious primary sequence homology between theTo antigen binding sites in RNaseP RNA and 7-2/MRP RNA; however, these sequences are capable of assuming a similar secondary structure which corresponds to the recently proposed cage structure for RNaseP RNAs and 7-2/MRP RNA (Forster and Altman (1989) Cell 62: 407–409). These data are supportive of the idea that these two RNAs may have evolved from a common progenitor molecule.     

Receptor binding and pH stability — How influenza A virus hemagglutinin affects host-specific virus infection

Influenza A virus strains adopt different host specificities mainly depending on their hemagglutinin (HA) protein. Via HA, the virus binds sialic acid receptors of the host cell and, upon endocytic uptake, HA triggers fusion between the viral envelope bilayer and the endosomal membrane by a low pH-induced conformational change leading to the release of the viral genome into the host cell cytoplasm. Both functions are crucial for viral infection enabling the genesis of new progeny virus.     

Meiosis Aspects and Nucleolar Activity in Triatoma vitticeps (Triatominae, Heteroptera)

Some aspects of both the nucleolar organizer activity and meiosis were studied in the testes of Triatoma vitticeps (Heteroptera, Triatominae). The techniques used included squashing followed by lacto-acetic orcein staining, silver-ion impregnation, fluorescent banding (CMA₃, Quinacrine mustard and DAPI) and fluorescent in situ hybridization (FISH). A close relationship between heterochromatin and nucleolus in testicular cells was observed. During meiosis, the silver-ion impregnation pattern varied. At metaphase plate, a small body appeared apart from the chromosomes. In the spermatids this small body was seen in preparations stained with orcein and silver- ion impregnation but not with fluorochromes or FISH. These characteristics combined suggest that these corpuscles represent a source of ribonucleoproteins (RNP) – RNA and specific nucleolar proteins. Silver-ion impregnation and (FISH) revealed nucleolar organizer activity in two metaphase sex chromosomes (X). These results indicate that, in these species, nucleolar organizer regions (NORs) are located in the sex chromosomes, X chromosomes were CMA₃⁺ and Y chromosome was DAPI⁺.     

Similarity between nuclear matrix proteins of various cells revealed by an improved isolation method

Comparative analysis of nuclear matrix proteins by two-dimensional electrophoresis may be greatly impaired by copurifying cytoskeletal proteins. The present data show that the bulk of adhering cytofilaments may mechanically be removed by shearing of nuclei pretreated with vanadyl ribonucleoside complexes. Potential mechanisms of action not based on ribonuclease inhibition are discussed. To individually preserve the integrity of nuclear structures, we developed protocols for the preparation of nuclear matrices from three categories of cells, namely leukocytes, cultured cells, and tissue cells. As exemplified with material from human lymphocytes, cultured amniotic cells, and liver tissue cells, the resulting patterns of nuclear matrix proteins appeared quite similar. Approximately 300 spots were shared among the cell types. Forty-nine of these were identified, 21 comprising heterogeneous nuclear ribonucleoproteins. Heterogeneous nuclear ribonucleoproteins L and nuclear lamin B2 isoforms were identified by amino acid sequencing and mass spectrometry. However, individually expressed proteins, such as the proliferating cell nuclear antigen, also pertained following application of the protocols. Thus, enhanced resolution and comparability of proteins improve systematic analyses of nuclear matrix proteins from various cellular sources. J. Cell. Biochem. 71:363–374, 1998. © 1998 Wiley-Liss, Inc.     

An SRp75/hnRNPG complex interacting with hnRNPE2 regulates the 5' splice site of tau exon 10, whose misregulation causes frontotemporal dementia

Tau is a neuronal-specific microtubule-associated protein that plays an important role in establishing neuronal polarity and maintaining the axonal cytoskeleton. Aggregated tau is the major component of neurofibrillary tangles (NFTs), structures present in the brains of people affected by neurodegenerative diseases called tauopathies. Tauopathies include Alzheimer's disease (AD), frontotemporal dementia with Parkinsonism (FTDP-17), the early onset dementia observed in Down syndrome (DS; trisomy 21) and the dementia component of myotonic dystrophy type 1 (DM1). Splicing misregulation of adult-specific exon 10, which codes for a microtubule binding domain, results in expression of abnormal ratios of tau isoforms, leading to FTDP-17. Positions 3 to 19 of the intron downstream of exon 10 define a hotspot of splicing regulation: the region diverges between humans and rodents, and point mutations within it result in tauopathies. In this study, we investigated three regulators of exon 10 splicing: serine/arginine-rich protein SRp75 and heterogeneous nuclear ribonucleoproteins hnRNPG and hnRNPE2. SRp75 and hnRNPG inhibit splicing of exon 10 whereas hnRNPE2 activates it. Using co-transfections, co-immunoprecipitations and RNAi we discovered that SRp75 binds to the proximal downstream intron of tau exon 10 at the FTDP-17 hotspot region; and that hnRNPG and hnRNPE2 interact with SRp75. Thus, increased exon 10 inclusion in FTDP mutants may arise from weakened SRp75 binding. This work provides insights into the splicing regulation of the tau gene and into possible strategies for correcting the imbalance in tauopathies caused by changes in the ratio of exon 10.     

Engineered virus-like particles for efficient in vivo delivery of therapeutic proteins

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Effective solvent systems for separation and an improved detection method for amino acids by paper chromatoghraphy

Binding of poly(A)-containing RNP to oligo(dT)-cellulose has been investigated as a function of mono- and divalent ion concentration. 80–90% binding was obtained either in high (500 mM) or in moderate NaCl concentrations in the presence of 5 mM MgCl₂. At 40 mM NaCl and 5 mM MgCl₂ poly(A)⁺-RNP exhibit approximately t he same stability as poly(A)⁺-RNA in binding to oligo(dT)-cellulose with a melting temperature of 41 and 45°C, respectively, indicating that the protein moeity has no effect on the ribonucleoprotein binding in these conditions. Differences were observed int he elution of poly(A)⁺-RNA and poly(A)⁺-RNP from oligo(dT)-cellulose in buffer without salts. Poly(A)⁺-RNA was completely removed at 4°C whereas the melting temperature of poly(A)⁺-RNP was only decreased to 34°C. The isolation of poly(A)⁺-RNP by thermal elution from oligo(dT)-cellulose is described.     

Purification of p53/55 kinase from nuclear ribonucleoproteins of Namalwa cells

Summary A nuclear p53/55 protein kinase has been isolated from nuclear ribonucleoprotein particles from human tumor cells. The enzyme was purified approximately 2200-fold from cell nuclei by sequential ribonuclease digestion of the RNP particles, DEAE cellulose and phosphocellulose chromatography. The kinase which was CAMP independent, catalyzed the phosphorylation of rabbit muscle glycogen synthase in the amino terminal domain, and conversion of the I to D form. The D synthase had a phosphorylation stoichiometry of 8 moles ³²P, per mole of synthase subunit with maximal specificity for ATP as phosphate donor; its K_m was 30 M. An antinucleolar antibody inhibited enzyme activity by 80%. Substrates for most other kinases were inactive. The kinase was essentially unaffected by the Walsh inhibitor, EGTA, regulatory subunits of protein kinase, calmodulin, trifluoperazine or heparin. Its activity was lost at 1 mM polyamine, but was enhanced 3-fold by MnC12 and 4- to 9-fold by deoxymononucleotides.The nuclei of HeLa cells contained 64% of the total kinase of which 11% were in nucleoli; the specific activity of the nucleolar kinase was twice that of the nuclear supernatant and four times that of the cytoplasmic kinase. These results indicate that nucleolar ribonucleoprotein particles of human tumor cells contain a CAMP-independent protein kinase which is similar to glycogen synthase kinase.Abbreviations RNP ribonucleoprotein - PMSF phenyl methyl sulfonyl fluoride - EGTA ethylene glycol-bis-(-aminoethyl ether) N,N-tetra acetic acid - TCA trichloroacetic acid - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - ELISA enzyme-linked immunosorbent assay