Guide RNA-directed uridine insertion RNA editing in vitro

Guide RNAs (gRNAs) have been proposed to mediate uridine (U) addition/deletion editing of mitochondrial mRNAs in kinetoplastid protozoa. The Us are proposed to be derived either from UTP by two successive cleavage-ligations or transesterifications, or from the 3' end of the gRNA by the same mechanisms. We have demonstrated gRNA-dependent U insertions into a specific editing site of a pre-edited mRNA which was incubated in a mitochondrial extract from Leishmania tarentolae. The predominant number of U insertions was determined by the number of guiding nucleotides in the added gRNA, and the formation of a gRNA-mRNA anchor duplex was necessary for activity. UTP and alpha-beta bond hydrolysis of ATP were required, and the activity was inhibited above 50-100 mM KCl. A gRNA-independent insertion of up to approximately 13 Us occurred in the absence of the added cognate gRNA; the extent of this activity was affected by sequences upstream and downstream of the edited region. Heparin inhibited the gRNA-independent U insertion activity and had no effect on the gRNA-dependent activity. Blocking the 3' OH of the gRNA had little effect on the gRNA-dependent U insertion activity. The data are consistent with a cleavage-ligation model in which the Us are derived directly from UTP.     

Messenger ribonucleic acid metabolism in mammalian mitochondria. Discrete poly(adenylic acid) lacking messenger ribonucleic acid species associated with mitochondrial polysomes

The mRNA species released from mitochondrial polysomes prepared by the Mg2+ precipitation technique have been further characterized using various analytical techniques. Mitochondrial polysomes were dissociated by treatment with puromycin and chemically labeled with (3H) dimethyl sulfate. About 51% of steady-state mitochondrial mRNA bind to oligo(dT)-cellulose indicating the presence of poly(adenylic acid)(poly(A)) in this fraction. The poly(A)-containing mRNAs resolve into discrete bands of 9-16 Se, while the RNA fraction unable to bind to oligo(dT)-cellulose representing poly(A)-lacking mRNA contains 8-12 Se species. About 90% of poly(A) lacking RNA hybridizes with mitochondrial DNA and less than 7% hybridizes with nuclear DNA. The extent of hybridization of poly(A)-lacking RNA with mitochondrial DNA was not significantly affected by the presence of excess mitochondrial rRNA, cytoplasmic rRNA, or a tenfold concentration of poly(A)-containing RNA isolated from total mitochondrial RNA. Possible differences in sequence properties between poly(A)-containing and -lacking mitochondrial mRNAs were further verified using a solid phase-bound cDNA procedure. Poly(A)-containing mRNA released from mitochondrial polysomes shows over 85% sequance homology with oligo(dT)-cellulose-bound cDNA prepared against total mitochondrial poly(A)-lacking mitochondrial mRNA hybridizes with the cDNA providing direct evidence for the distinct sequence properties of the two mRNA species.     

Association of a group I intron with its splice junction in 50S ribosomes: implications for intron toxicity

The effect of genetic context on splicing of group I introns is not well understood at present. The influence of ribosomal RNA conformation on splicing of rDNA introns in vivo was investigated using a heterologous system in which the Tetrahymena group I intron is inserted into the homologous position of the Escherichia coli 23S rRNA. Mutations that block splicing in E. coli result in accumulation of unspliced 23S rRNA that is assembled into 50S complexes, but not 70S ribosomes. The data indicate that accommodation of the intron structure on the surface of the 50S subunit inhibits interactions with the small ribosomal subunit. Spliced intron RNA also remains noncovalently bound to 50S subunits on sucrose gradients. This interaction appears to be mediated by base pairing between the intron guide sequence and the 23S rRNA, because the fraction of bound intron RNA is reduced by point mutations in the IGS or deletion of the P1 helix. Association of the intron with 50S subunits correlates with slow cell growth. The results suggest that group I introns have the potential to inhibit protein synthesis in prokaryotes by direct interactions with ribosomes.     

A 68-nucleotide sequence within the 3' noncoding region of simian hemorrhagic fever virus negative-strand RNA binds to four MA104 cell proteins

The 3' noncoding region (NCR) of the negative-strand RNA [3'(-)NCR RNA] of the arterivirus simian hemorrhagic fever virus (SHFV) is 209 nucleotides (nt) in length. Since this 3' region, designated 3'(-)209, is the site of initiation of full-length positive-strand RNA and is the template for the synthesis of the 5' leader sequence, which is found on both full-length and subgenomic mRNAs, it is likely to contain cis-acting signals for RNA synthesis and to interact with cellular and viral proteins to form replication complexes. Gel mobility shift assays showed that cellular proteins in MA104 S100 cytoplasmic extracts formed two complexes with the SHFV 3'(-)209 RNA, and results from competition gel mobility shift assays demonstrated that these interactions were specific. Four proteins with molecular masses of 103, 86, 55, and 36 kDa were detected in UV-induced cross-linking assays, and three of these proteins (103, 55, and 36 kDa) were also detected by Northwestern blotting assays. Identical gel mobility shift and UV-induced cross-linking patterns were obtained with uninfected and SHFV-infected extracts, indicating that the four proteins detected are cellular, not viral, proteins. The binding sites for the four cellular proteins were mapped to the region between nt 117 and 184 (68-nt sequence) from the 3' end of the SHFV negative-strand RNA. This 68-nt sequence was predicted to form two stem-loops, SL4 and SL5. The 3'(-)NCR RNA of another arterivirus, lactate dehydrogenase-elevating virus C (LDV-C), competed with the SHFV 3'(-)209 RNA in competition gel mobility shift assays. UV-induced cross-linking assays showed that four MA104 cellular proteins with the same molecular masses as those that bind to the SHFV 3'(-)209 RNA also bind to the LDV-C 3'(-)NCR RNA and equine arteritis virus 3'(-)NCR RNA. However, each of these viral RNAs also bound to an additional MA104 protein. The binding sites for the MA104 cellular proteins were shown to be located in similar positions in the LDV-C 3'(-)NCR and SHFV 3'(-)209 RNAs. These data suggest that the binding sites for a set of the cellular proteins are conserved in all arterivirus RNAs and that these cell proteins may be utilized as components of viral replication complexes.     

A three-dimensional working model for a guide RNA from Trypanosoma brucei

RNA editing in protozoan parasites is a mitochondrial RNA processing reaction in which exclusively uridylate residues are inserted into, and less frequently deleted from, pre-mRNAs. Molecules central to the process are so-called guide RNAs (gRNAs) which function as templates in the reaction. For a detailed molecular understanding of the mechanism of the editing process knowledge of structural features of gRNAs will be essential. Here we report on a computer-assisted molecular modelling approach to construct the first three-dimensional gRNA model for gND7-506, a ND7-specific gRNA from Trypanosoma brucei. The modelling process relied on chemical modification and enzymatic probing data and was validated by in vitro mutagenesis experiments. The model predicts a reasonably compact structure, where two stem/loop secondary structure elements are brought into close proximity by a triple A tertiary interaction, forming a core element within the centre of the molecule. The model further suggests that the surface of the gRNA is primarily made up of the sugar-phoshate backbone. On the basis of the model, footprinting experiments of gND7-506 in a complex with the gRNA binding protein gBP21 could successfully be interpreted and provide a first picture for the assembly of gRNAs within a ribonucleoprotein complex.     

Ectopic pairing and evolution of 5S ribosomal RNA genes in the chromosomes of Drosophila funebris

5S ribosomal RNA from Drosophila melanogaster labeled with 125I was used to locate the 5S rRNA genes in chromosomes of D. funebris by means of in situ hybridization. Silver grains were observed at three distinct sites, one of which was a recognized reverse repeat. Only one half of the reverse repeat, however, hybridizes with 5S rRNA and the significance of this phenomenon is discussed. A case of ectopic pairing between two different 5S sites in the genome is reported, and the significance of ectopic pairing is considered.     

A novel DNA-peptide complex for efficient gene transfer and expression in mammalian cells

To develop a nonviral gene delivery system for treatment of diseases, our strategy is to construct DNA complexes with short synthetic peptides that mimic the functions of viral proteins. We have designed and synthesized two peptides which emulate viral functions - a DNA condensing agent, YKAK(8)WK, and an amphipathic, pH-dependent endosomal releasing agent, GLFEALLELLESLWELLLEA. The active gene delivery complex was constructed step-wise through a spontaneous self-assembly process involving oppositely charged, electrostatic interactions. To assemble DNA-peptide complexes with different overall net charges, only the negative charges of DNA phosphate, the positive charges of the 10 epsilon-amino groups of YKAK(8)WK and the negative charges of the 5 gamma-carboxyl groups of GLFEALLELLESLWELLLEA were considered. In the first step, negatively charged DNA was rapidly-mixed with an excess of YKAK(8)WK to form positively charged DNA-YKAK(8)WK complexes, which gave little gene transfer. In the second step and to form the active complex,the cationic DNA complex was rapidly mixed with spontaneously incorporated through electrostatic interactions. Transfection using these complexes of CMV-luc, YKAK(8)WK and GLFEALLELLESLWELLLEA gave high-levels of gene expression in a variety of cell lines. These simple DNA complexes, which contain only three molecularly defined components, have general utility for gene delivery and can replace viral vectors and cationic lipids for some applications in gene therapy.     

Living-related donor liver transplantation: status quo in Kyoto, Japan

Recent studies have implicated nucleotides in diverse and unexpected functions related to p53 levels, p53-dependent G0/G1 cell cycle arrest, and the role of dATP in the activation of the caspase-induced apoptosis. Using deoxyadenosine-resistant L1210 cells (ED2 and Y8) that had ribonucleotide reductase that was not sensitive to inhibition by dATP and also exhibited other metabolic alterations, the properties of these cells with respect to the role(s) of nucleotides in these functions were explored. In the ED2 and Y8 cells that did not express p53 protein, the pools of UTP, CTP, ATP, and GTP were markedly decreased. The decreased cellular levels of UTP and CTP did not result in these cells being more sensitive to either PALA or acivicin. The ED2 and Y8 cells did not block in G0/G1 in response to PALA treatment even though the basal cellular concentrations of UTP and CTP were reduced 50 to 80%. While it has been shown that dATP in combination with cytochrome c is involved in the apoptotic pathway, the concentration of exogenous deoxyadenosine required to induce apoptosis in the parental L1210 cells was far in excess of the concentration required to inhibit cell growth. Deoxyadenosine did not cause an increase in apoptosis in the deoxyadenosine-resistant Y8 cells. These data suggest that the new roles ascribed to nucleotides may be specific for the particular cell type under very specific conditions.     

Dimerization of MoMuLV genomic RNA: redefinition of the role of the palindromic stem-loop H1 (278-303) and new roles for stem-loops H2 (310-352) and H3 (355-374)

Genomic RNAs from retroviruses are packaged as dimers of two identical RNA molecules. In Moloney murine leukemia virus, a stem-loop structure (H1) located in the encapsidation domain Psi (nucleotides 215-564) was postulated to trigger RNA dimerization through base pairing between auto complementary sequences. The Psi domain also contains two other stem-loop structures (H2 and H3) that are essential for RNA packaging. Since it was suspected than H1 is not the only element involved in RNA dimerization, we systematically investigated the dimerization capacity of several subdomains of the first 725 nucleotides of genomic RNA. The efficiency of dimerization of the various RNAs was estimated by measuring their apparent dissociation constants, and the specificity was tested by competition experiments. Our results indicate that the specificity of dimerization of RNA nucleotides 1-725 is driven by motifs H1-H3 in domain Psi. To define the relative contributions of these elements, RNA deletion mutants containing different combinations of H1-H3 were constructed and further analyzed in competition and kinetic experiments. Our results confirm the importance of H1 in triggering dimerization and shed new light on the mechanism of dimerization. H1 is required to provide a stable dimer, probably through the formation of extended intermolecular interactions. However, H1-mediated association is a slow process that is kinetically enhanced by H3, and to a lesser extent by H2. We suggest that they facilitate the recognition between the two RNAs, most likely through their conserved GACG loops. Our results reinforce the idea that dimerization and packaging are two closely related processes.     

Amino acid sequence of a four-iron-four-sulphur ferredoxin isolated from Bacillus stearothermophilus

A novel method of RNA fractionation based on a gradual release of the RNA molecules from ribonucleoprotein complexes has been used for the analysis of ribosomal and non-ribosomal complexes of rat liver cytoplasm. Adsorption of native ribonucleoproteins on a Celite column (occuring through only the protein moiety) followed by a consequent dissociation of RNP complexes brought about by various agents results in RNA fractionation in accordance with the tightness of the RNA-protein bonds. The cytoplasmic ribosomal and rapidly labelled non-ribosomal RNA species are separated into several fractions identified as 18S and 28S rRNA's, mRNA and messenger-like RNA. A relatively small fraction (about 10% of the total) of rRNA tenaciously bound to protein has been also revealed.