Compartmentation of uridine 5′-triphosphate occurs during synthesis of cytoplasmic and mitochondrial ribosomal RNAs of cultured tomato cells but not during synthesis of cytoplasmic ribosomal and transfer RNAs

Compartmentation of uridine 5-triphosphate (UTP) was studied during the nucleolar synthesis of cytoplasmic ribosomal RNA (cyt-rRNA) and the synthesis of cytoplasmic transfer RNA (cyt-tRNA) in the nuclear matrix as well as the synthesis of mitochondrial ribosomal RNA (mt-rRNA) in tomato (Lycopersicon esculentum Mill. cv. Lukullus) cell-suspension culture using the approach of Wiegers et al. (Eur. J. Biochem. 64, 535–540, 1976). Before measurements were made, it was ensured that: (i) there was steady-state labeling of all RNAs studied as well as UTP; (ii) there was stability of cyt-tRNA and cyt-rRNA; (iii) there was no label randomization through degradation of [³H]uridine; (iv) there were significant differences in the specific radioactivity of UTP, the final immediate precursor of RNA, after supplying the cells with two different exogenous [³H]uridine concentrations.By comparing the steady-state specific radioactivity of UTP with that of cyt-tRNA and cyt-18S rRNA during constant [³H]uridine supply, we found that the three molecules had equal specific radioactivities which, however, differed significantly from that of the mt-rRNA. With a 20-fold higher uridine concentration, i.e. a 20-fold lower specific radioactivity of exogenous [³H]uridine, the specific radioactivity of cyt-rRNA, cyt-tRNA and UTP decreased proportionally whereas that of mt-RNA increased. These results argue against different UTP pools during synthesis of cyt-rRNA and cyt-tRNA, but indicate compartmentation of UTP during rRNA synthesis in the nucleus and the mitochondria of tomato cells.Abbreviations CMP cytidine 5-monophosphate - cyt-rRNA cytoplasmic ribosomal RNA - cyt-tRNA cytoplasmic transfer RNA - mt-rRNA mitochondrial rRNA - NC nitrocellulose - PAGE polyacrylamide gel electrophoresis - TLC thin-layer chromatography - Tris 2-amino-2-(hydroxymethyl)-1,3-propanediol - UDP uridine 5-diphosphate - UMP uridine 5-monophosphate - UTP uridine 5-triphosphate     

Rates of synthesis of major classes of RNA in Drosophila embryos.

We have been successful in labeling to high specific activity (3 × 10⁵ dpm/μg) the RNA synthesized by large numbers of Drosophila embryos. Embryos of various developmental stages were rendered permeable with octane and labeled with [³H]uridine for 1 hr. At each stage the total dpm incorporated into RNA and the specific activity of the UTP pool were measured and used to calculate the absolute rate of RNA synthesis per embryo. This rate increases during embryonic development, from 1 pmole UTP/hr at 2 hr after oviposition to 6 pmoles UTP/hr at 15 hr. The rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNAs were determined by analyzing the fractionated RNAs from each stage by sucrose gradient sedimentation. There is a significant activation of nuclear RNA synthesis at the blastoderm stage (approximately 2 hr after oviposition). After blastoderm, the rates of synthesis of nuclear and cytoplasmic poly(A)^? and poly(A)⁺ RNA per embryo increase continuously; the rate of synthesis of each of these classes per nucleus, however, remains fairly constant. After making corrections for turnover during the labeling period, we find that the rates of synthesis of the major classes of RNA per nucleus at the gastrula stage are: cytoplasmic poly(A)⁺ RNA, 0.06 fg/nucleus-min; hnRNA, 0.86 fg/nucleus-min; and ribosomal RNA, 0.46 fg/nucleus-min. These rates are compared to rates of RNA synthesis in sea urchin embryos.     

Effect of cytokinins on ribosomal RNA gene expression in excised cotyledons of Cucurbita pepo L.

Excised pumpkin (Cucurbita pepo L.) cotyledons were used to investigate the effects of two different types of cytokinins: N⁶-benzyladenine and N₁-(2-chloro-4-pyridyl)-N₂-phenylurea on RNA synthesis in isolated nuclei. Treatment of cotyledons with both cytokinins resulted in a rapid enhancement of nuclear RNA-polymerase-I activity (EC 2.7.7.6). Maximum stimulation of RNA polymerase I, responsible for rRNA synthesis, was observed 4–6 h after the start of cytokinin action. The activity of RNA polymerase II was stimulated much more slowly and to a lesser extent. Uridine 5-monophosphate-uridine analysis of the alkalidigested nascent pre-rRNA chains showed that the stimulation of RNA-polymerase-I activity was the consequence of an increase of the polyribonucleotide-clongation rate. No significant change in the number of transcribing enzyme molecules was defected after hormone treatment (86·10³ RNA-polymerase-I molecules per diploid genome).Indications that de-novo protein synthesis is necessary for cytokinin-mediated RNA-polymerase stimulation were derived from experiments showing inhibition by cycloheximide.Abbreviations BA N⁶-benzyladenine - [PU]-30 N₁-(2-chloro-4-pyridyl)-N₂-phenylurea - UMP undine 5-monophosphate - UTP udine 5-triphosphate     

Rates of RNA synthesis during early embryogenesis in Drosophila melanogaster

We determined the absolute rates of RNA synthesis during embryogenesis in Drosophila melanogaster by measuring the incorporation of ³H-5-orotic acid into RNA, and the specific activity of the UTP pool. Initially (preblastoderm) the rate of RNA synthesis is relatively high, but declines to a lower level by gastrulation. The data suggest that RNA synthesis is initiated during very early embryogenesis.     

Double-stranded RNA replicons associated with chloroplasts of a green alga,Bryopsis cinicola

Double-stranded RNAs (dsRNAs) associated with chloroplasts and mitochondria have been found in the coenocytic green alga Bryopsis cinicola. In this study we report molecular properties of the four chloroplast-associated dsRNAs (BDRC1 to BDRC4) The longest dsRNA molecule (BDRC1) was sequenced entirely (1959 bp) and a single large ORF of 1722 bp was found within it. Database searches revealed similarities between the deduced amino acid sequence of this ORF and RNA-dependent RNA polymerase (RdRp) sequences from several RNA viruses. The most similar sequence in the database was the RdRp of beet cryptic virus 3. Phylogenetic analysis revealed that the RdRp-like sequence of BDRC1 can be placed in the Partitiviridae clade. To detect autonomous replication of these dsRNAs, RdRp assays were carried out with actinomycin D, which is an inhibitor of DNA-dependent RNA synthesis. Incorporation of [-³²P]UTP was detected specifically in the chloroplast and mitochondrial dsRNAs, indicating that both the chloroplast dsRNAs (BDRCs) and the mitochondrial dsRNA (BDRM) of B. cinicola are RNA replicons. The green alga B. cinicola harbors different dsRNA replicons in its chloroplasts and mitochondria.     

RNA turnover and protein synthesis in fish cells

Protein synthesis in fish has been previously correlated with RNA content. The present study investigates whether protein and RNA synthesis rates are similarly related. Protein and RNA synthesis rates were determined from ³H-phenylalanine and ³H-uridine incorporation, respectively, and expressed as % · day⁻¹ and half-lives, respectively. Three fibroblast cell lines were used: BF-2, RTP, CHSE 214, which are derived from the bluegill, rainbow trout and Chinook salmon, respectively. These cells contained similar RNA concentrations (∼175 μg RNA · mg⁻¹ cell protein). Therefore differences in protein synthesis rates, BF-2 (31.3 ± 1.8)>RTP (25.1 ± 1.7)>CHSE 214 (17.6 ± 1.1), were attributable to RNA translational efficiency. The most translationally efficient RNA (BF-2 cells), 1.8 mg protein synthesised · μg⁻¹ RNA · day⁻¹, corresponded to the lowest RNA half-life, 75.4 ± 6.4 h. Translationally efficient RNA was also energetically efficient with BF-2 cells exploiting the least costly route of nucleotide supply (i.e. exogenous salvage) 3.5–6.0 times more than the least translationally efficient RNA (CHSE 214 cells). These data suggest that differential nucleotide supply, between intracellular synthesis and exogenous salvage, constitutes the area of pre-translational flexibility exploited to maintain RNA synthesis as a fixed energetic cost component of protein synthesis. Accepted: 12 November 1999     

Nucleotide pools of Novikoff rat hepatoma cells growing in suspension culture. II. Independent snucleotide pools for nucleic acid synthesis

Novikoff rat hepatoma cells (subline NlSl-67) in suspension culture incorporate ³H-5-uridine into the acid-soluble nucleotide pool more rapidly than into RNA, resulting in the accumulation of labeled UTP in the cells. When labeled uridine is removed from the medium after 20 minutes or 4.75 hours of labeling, the rate of incorporation of label from the nucleotide pool into RNA decreases to less than 10% of the original rate within five to ten minutes, in spite of the presence of a large pool of labeled UTP in the cells, and incorporation ceases completely if an excess of unlabeled uridine is present during the chase. Upon addition of ¹⁴C-uridine to ³H-uridine pulse-labeled, chased cells, the ¹⁴C begins to be incorporated into RNA without delay and at a rate predetermined by the concentration of ¹⁴C-uridine in the medium and without affecting the fate of the free ³H-nucleotides labeled during the pulse-period. The results are interpreted to indicate that uridine is incorporated into at least two different pools, only one of which serves as primary source of nucleotides for RNA synthesis. During active synthesis of RNA, the latter pool of free nucleotides is very small and rapidly exhausted when uridine is removed from the medium. However, UTP accumulates in this pool when cells are labeled at 4–6°, since at this temperature RNA synthesis is blocked while uridine is still phosphorylated by the cells, and the UTP is rapidly incorporated into RNA during a subsequent ten-minute chase at 37°. From these types of experiments it is estimated that only 20–25% of the total uridine nucleotides formed in the cells from uridine in the medium is directly available for RNA synthesis and that the remainder becomes available only at a slow rate. Evidence is presented which suggests that one uridine nucleotide pool is located in the cytoplasm and another in the nucleus and that mainly the nuclear pool supplies nucleotides for RNA synthesis. The size of the latter pool is under strict regulatory control, since preincubation of the cells with 0.5 mM unlabeled uridine has little or no effect on the subsequent incorporation of ³H-uridine, although it results in an increase of the overall cellular uridine nucleotide content to at least 5 mM. Other results indicate that adenosine is also incorporated into two independent nucleotide pools, whereas the cells normally appear to possess a single thymidine nucleotide pool.     

Mechanism of U-Insertion RNA Editing in Trypanosome Mitochondria: Characterization of RET2 Functional Domains by Mutational Analysis

3′-Terminal uridylyl transferases (TUTases) selectively bind uridine 5′-triphosphate (UTP) and catalyze the addition of uridine 5′-monophosphate to the 3′-hydroxyl of RNA substrates in a template-independent manner. RNA editing TUTase 1 and RNA editing TUTase 2 (RET2) play central roles in uridine insertion/deletion RNA editing, which is an essential part of mitochondrial RNA processing in trypanosomes. Although the conserved N-terminal (catalytic) domain and C-terminal (nucleotide base recognition) domain are readily distinguished in all known TUTases, nucleotide specificity, RNA substrate preference, processivity, quaternary structures, and auxiliary domains vary significantly among enzymes of divergent biological functions. RET2 acts as a subunit of the RNA editing core complex to carry out guide-RNA-dependent U-insertion into mitochondrial mRNA. By correlating mutational effects on RET2 activity as recombinant protein and as RNA editing core complex subunit with RNAi-based knock-in phenotypes, we have assessed the UTP and RNA binding sites in RET2. Here we demonstrate functional conservation of key UTP-binding and metal-ion-coordinating residues and identify amino acids involved in RNA substrate recognition. Invariant arginine residues 144 and 435 positioned in the vicinity of the UTP binding site are critical for RET2 activity on single-stranded and double-stranded RNAs, as well as function in vivo. Recognition of a double-stranded RNA, which resembles a guide RNA/mRNA duplex, is further facilitated by multipoint contacts across the RET2-specific middle domain.     

Poliovirus cis-acting replication element-dependent VPg Uridylylation lowers the Km of the initiating nucleoside triphosphate for viral RNA replication

Initiation of RNA synthesis by RNA-dependent RNA polymerases occurs when a phosphodiester bond is formed between the first two nucleotides in the 5′ terminus of product RNA. The concentration of initiating nucleoside triphosphates (NTPi) required for RNA synthesis is typically greater than the concentration of NTPs required for elongation. VPg, a small viral protein, is covalently attached to the 5′ end of picornavirus negative- and positive-strand RNAs. A cis-acting replication element (CRE) within picornavirus RNAs serves as a template for the uridylylation of VPg, resulting in the synthesis of VPgpUpU_OH. Mutations within the CRE RNA structure prevent VPg uridylylation. While the tyrosine hydroxyl of VPg can prime negative-strand RNA synthesis in a CRE- and VPgpUpU_OH-independent manner, CRE-dependent VPgpUpU_OH synthesis is absolutely required for positive-strand RNA synthesis. As reported herein, low concentrations of UTP did not support negative-strand RNA synthesis when CRE-disrupting mutations prevented VPg uridylylation, whereas correspondingly low concentrations of CTP or GTP had no negative effects on the magnitude of CRE-independent negative-strand RNA synthesis. The experimental data indicate that CRE-dependent VPg uridylylation lowers the K_m of UTP required for viral RNA replication and that CRE-dependent VPgpUpU_OH synthesis was required for efficient negative-strand RNA synthesis, especially when UTP concentrations were limiting. By lowering the concentration of UTP needed for the initiation of RNA replication, CRE-dependent VPg uridylylation provides a mechanism for a more robust initiation of RNA replication.