A small nuclear precursor of messenger RNA in the cellular slime mold Dictyostelium discoideum

Previous work (Firtel et al., 1972) showed that messenger RNA from the cellular slime mold Dictyostelium discoideum, like that from mammalian cells, contains a sequence of about 100 adenylic acid residues at the 3′ end. We show here that Dictyostelium nuclei, labeled under a variety of conditions, do not contain material analogous to the large nuclear heterogeneous RNA found in mammalian cells. Rather, the majority of pulse-labeled nuclear RNA that is not a precursor of ribosomal RNA does contain at least one sequence of polyadenylic acid; this RNA, with an average molecular weight of 500,000, appears to be only 20% larger than cytoplasmic messenger RNA.Pulse-labeling experiments show that the nuclear poly(A)-containing RNA is a material precursor of messenger RNA. Whereas previous work showed that over 90% of messenger RNA sequences are transcribed from non-reiterated DNA, we show here that about 25% of nuclear poly (A)-containing RNA is transcribed from reiterated DNA sequences and only 75% from single-copy DNA. We present evidence that a large fraction of the nuclear poly(A)-containing RNA contains, at the 5′ end, a sequence of about 300 nucleotides that is transcribed from repetitive DNA, and which is lost before transport of messenger RNA into the cytoplasm.Based on these and other results, we present a model of arrangement of repetitive and single-copy DNA sequences in the Dictyostelium chromosome.     

5'-terminal phosphorylation and secondary structure of double-stranded RNA from a fungal virus.

Different double-stranded RNA species from Penicillium stoloniferum virus have been phosphorylated at the 5′ termini with the aid of polynucleotide kinase. A very low phosphate uptake has been observed which, especially in the case of a relatively small molecular component, was increased several times by pretreatment with RNAase t₁. Adenosine and uridine have been detected at the 5′-termini of this RNA component. Digestion with RNAase T₁, an enzyme which does not cut across the two strands of a double-stranded RNA molecule, produced a new uridine terminus and increased the efficiency of phosphorylation. It is concluded that this double-stranded RNA molecule contains single-stranded stretches at or near the 5′-termini. The possibility of a circular structure being formed by the annealing of single-stranded tails is discussed.     

Methylation of Sindbis virus "26S" messenger RNA.

The main virus-specific messenger RNA species of Sindbis virus-infected hamster cells, the “26S” RNA, has been examined with regard to methylation status. Internal methylated residues and terminal methylated residues were present, in approximately equal amounts. The internal methyl groups were almost all in 5-methylcytosine residues and the terminal methyl groups were mainly in 7-methylguanine residues. Evidence is presented that these latter occur in “capped” 5′-termini with the novel structure m⁷G(5′)pppNp.     

Oligo(2′-<Emphasis Type="Italic">O</Emphasis>-methylribonucleotides) and their derivatives: III. 5′-Mono- and 5′-bispyrenyl derivatives of oligo(2′-<Emphasis Type="Italic">O</Emphasis>-methylribonucleotides) and their 3′-modified analogues: Synthesis and properties

5′-Pyrenylmethylphosphamide and 5′-bispyrenylmethylphosphordiamide derivatives of oligo(2′-O-methylribonucleotides) and their analogues with thymidine attached at their 3′-termini by a 3′-3′-phosphodiester internucleotide bond (“inverted” thymidine) were synthesized. The effect of the pyrene residue(s) on the thermal stability of duplexes of the modified oligonucleotides with RNA and DNA was studied. A possibility of detection of hybridization of 5′-mono- and 5′-bispyrenyl derivatives with RNA and DNA targets in solution was demonstrated according to the changes in fluorescence. 5′-Pyrenylphosphamide derivatives of oligo(2′-O-methylribonucleotides) and their inverted analogues were shown to be used as sensitive probes for the detection of single nucleotide polymorphisms in RNA and DNA by the method of thermal duplex denaturation with fluorescence change registration.     

Identification of a high molecular weight presumptive precursor to albumin mRNA in the nucleus of rat liver and hepatoma cell line H4AZC2.

Nuclear RNAs prepared from rat liver and rat hepatoma cell line H4AZC2 have been fractionated and examined for albumin mRNA sequences by annealing to specific albumin [3H]cDNA. In both instances, sucrose gradient analysis revealed nuclear RNA molecules containing albumin RNA sequences which sedimented at 26 S (26 S albumin RNA). In contrast, cytoplasmic albumin messenger RNA sediments exclusively at 17 S. 26 S albumin RNA is resistant to both heat denaturation (65 degrees C X 5 min) and denaturation in 85% formamide (v/v), and 75% of these molecules are polyadenylated. These results provide evidence for the existence of an intact, high molecular weight, polyadenylated nuclear RNA which contains albumin mRNA sequences.     

Cytoplasmic methylation of mature 5.8 S ribosomal RNA

Levels of 2-O-methylation were determined in ribosomal 5·8 S RNAs from whole cells and both the nuclear and cytoplasmic fractions of rat liver, rat kidney cells in culture (NRK) and HeLa cells. All 5·8 S RNA molecules contained the alkali stable Gm-Cp dinucleotide at position 77 but only whole cell rat liver RNA contained large amounts (0·7 mol) of Um at position 14. All nuclear 5·8 S RNA fractions were largely undermethylated at this site. In contrast, cytoplasmic 5.8 S RNA from rat liver and, to a lesser degree, NRK cells contained significantly more Um; up to 80% of the molecules from rat liver contained the methylated residue. These results indicate that mature 5·8 S RNA can be methylated in the cytoplasm. When labeling kinetics were examined in NRK cells, the methylation at residue 14 was found to increase as a function of the time spent in the cytoplasm, confirming that this modification is, unlike other ribosomal RNA methylations, in part or largely cytoplasmic.     

The synthesis and processing of a nuclear RNA precursor to rat pregrowth hormone messenger RNA.

A recombinant DNA plasmid, pBR322-GH1, which contains about 80% of the sequences of rat pregrowth hormone (pGH) mRNA, allowed an analysis of nuclear RNA from GH3 cells for possible precursors of cytoplasmic pGH mRNA. A single 20-22S RNA SPECIES ABOUT 2-3 TIMes larger than pGH mRNA was detected in nuclear RNA from GH3 cells labeled for 5 min. with 3H-uridine. After longer label times a 12S RNA indistinguishable in size from cytoplasmic 12S pGH mRNA became the predominant labeled RNA complementary to the plasmid pBR322-GH1. Both of these nuclear RNA species contained poly (A). Kinetic analysis of the labeling of nuclear and cytoplasmic pGH mRNA sequences showed that the 20S and 12S nuclear RNA molecules were labeled before significant labeling of cytoplasmic pGH mRNA was detected, and also indicated that there is complete conservation of nuclear pGH mRNA sequences in the production of cytoplasmic pGH mRNA. These results indicate that cytoplasmic pGH mRNA is generated by nuclear processing of a larger nuclear RNA molecule.     

5'-terminal structures of poly(A)+ cytoplasmic messenger RNA and of poly(A)+ and poly(A)- heterogeneous nuclear RNA of cells of the dipteran Drosophila melanogaster.

Structures at the 5′ terminus of poly (A)-containing cytoplasmic RNA and heterogeneous nuclear RNA containing and lacking poly(A) have been examined in RNA extracted from both normal and heat-shocked Drosophila cells. ³²P-labeled RNA was digested with ribonucleases T₂, T₁ and A and the products fractionated by a fingerprinting procedure which separates both unblocked 5′ phosphorylated termini and the blocked, methylated, “capped” termini, known to be present in the messenger RNA of most eukaryotes.Approximately 80% of the 5′-terminal structures recovered from digests of poly(A)-containing Drosophila mRNA are cap structures of the general form m⁷G^5′ppp^5′X(m)pY(m)pZp. With respect to the extent of ribose methylation and the base distribution, the 5′-terminal sequences of Drosophila capped mRNA appear to be intermediate between those of unicellular eukaryotes and those of mammals. Drosophila is the first organism known in which type 0 (no ribose methylations), type 1 (one ribose methylation), and type 2 (two ribose methylations) caps are all present. In contrast to mammalian cells, the caps of Drosophila never contain the doubly methylated nucleoside N⁶,2′-O-dimethyladenosine. Both purines and pyrimidines can be found as the penultimate nucleoside of Drosophila caps and there is a wide variety of X-Y base combinations. The relative frequencies of these different base combinations, and the extent of ribose methylation, vary with the duration of labeling. The large majority of poly(A)-containing cytoplasmic RNA molecules from heat-shocked Drosophila cells are also capped, but these caps are unusual in having almost exclusively purines as the penultimate X base.Greater than 75% of the 5′ termini of heterogeneous nuclear RNA (hnRNA) containing poly(A) and greater than 50% of the termini of hnRNA lacking poly (A) are also capped. Triphosphorylated nucleotides, common as the 5′ nucleotides of mammalian hnRNA, are rare in the poly(A)-containing hnRNA of Drosophila. The frequency of the various type 0 and type 1 cap sequences of cytoplasmic and nuclear poly (A)-containing RNA are almost identical. The caps of hnRNA lacking poly(A) are also quite similar to those of poly-adenylated hnRNA, but are somewhat lower in their content of penultimate pyrimidine nucleosides, suggesting that these two populations of molecules are not identical.     

RNA metabolism in nuclei isolated from HeLa cells.

Nuclei with low cytoplasmic contamination, capable of synthesizing RNA for an extended period of time, were prepared from HeLa cells. Besides elongating RNA chains already initiated in vivo, the nuclear preparation initiates the synthesis of new RNA chains. This was shown by labelling the newly synthesized RNA with [gamma-32P]GTP and by detecting the presence of labelled guanosine tetraphosphate among the alkaline hydrolysis products of synthesized RNA. By synthesizing RNA in the presence of each of the four gamma-32P-labelled nucleoside triphosphates, it was possible to conclude that RNA chain synthesis starts predominantly with a purine base. Both nucleolar and nucleoplasmic RNAs are made. The nuclear preparation methylates the nucleolar RNA by utilizing S-adenosyl-L-methionine as a methyl-group donor.     

Acute effect of 5-fluorouracil on cytoplasmic and nuclear dihydrofolate reductase messenger RNA metabolism

Studies were completed in C3-L5178Y cells, in which the DNA-coding region for dihydrofolate reductase messenger RNA (DHFR-mRNA) is amplified, to determine the acute effect of 5-fluorouracil (FUra) on DHFR-mRNA metabolism. There was minimal to no effect of 100 microM FUra on total cytoplasmic DHFR-mRNA levels by 6 and 12 h and only a 25% reduction by 24 h. These results contrasted with the nuclear DHFR-mRNA levels which by 6 h following exposure to FUra increased by 80% in a dose-dependent manner. Furthermore, some of the increased nuclear DHFR-mRNA was found to be in a non-polyadenylated form. Under conditions to examine only RNA synthesized during the drug exposure, FUra was found to markedly enhance the level of newly synthesized nuclear DHFR-mRNA in a dose-dependent manner, while also producing an apparent dose-dependent reduction in the cytoplasmic DHFR-mRNA. RNA fractionated by 1.5% agarose-urea gel electrophoresis revealed two major cytoplasmic DHFR-mRNA species approximately 1.8 and 0.8 kilobases in size. Following a 24-h FUra exposure, a dose-dependent loss of the 0.8-kilobase DHFR-mRNA was observed. The combined results of these experiments indicate that FUra treatment reduces the ability of nascent DHFR-mRNA to relocate to the cytoplasm, suggesting either an inhibition of mRNA processing or nuclear-cytoplasmic transport.