Structure and metabolism of adenovirus RNAs containing sequences from the fiber gene

The body of adenovirus fiber messenger RNA is specified by viral r-strand co-ordinates 86.2 to 91.2. Since this mRNA is transcribed from the major late promoter at map position 16, nuclear precursors to the mRNA could be as large as 84% of the length of the 35,000 nucleotide genome. This study identified and characterized polyadenylated nuclear RNAs that contain fiber sequences and therefore are possible processing intermediates. These nuclear RNAs were characterized by hybridization of [³H]RNA preparations and by electron microscopy of RNA-DNA hybrids. Three size classes of RNAs containing fiber sequences were identified: (1) a 22 S species maps from 86.2 to 90.3. This RNA has essentially the same co-ordinates as fiber mRNA. (2) Two 28 S species have co-ordinates of 80.1 to 90.4 and 85.9 to 96.9, respectively. Thus one species has a 5′ terminus coincident with that of the mRNA body, and one has a 3′ terminus coincident with that of the 3′ end of the mRNA body. The polyadenylated terminus at 96.9 does not coincide with the 3′ end of any known mRNA. (3) There are at least two 35 S species. The 3′ end of one species is coincident with that of fiber mRNA. The 3′ terminus of the second RNA is at approximately 96.9.The labeling kinetics of each of these polyadenylated nuclear RNAs were investigated. In continuous label experiments, the two 35 S RNAs and the 85.9 to 96.9 28 S RNA became uniformly labeled in approximately 60 minutes. The 22 S RNA and the 80.1 to 90.4 28 S species continued to accumulate for at least several hours. These results are consistent with a precursor function for the 35 S RNAs and the 85.9 to 96.9 28 S species. The structures of the putative precursors imply that processing of the 3′ end is not a prerequisite for 5′ cleavage.     

Detection of cyclin D1 mRNA by hybridization sensitive NIC–oligonucleotide probe

A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2′-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2′-deoxy-uridine nucleoside, dU^TO, (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2′-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dU^TO at various positions. dU^TO-2′-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250 ng/μl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840 ng/μl). Additionally, dU^T can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dU^TO oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer.     

Processing of RNA in Escherichia coli is limited in the absence of ribonuclease III,ribonuclease E and ribonuclease P

A strain of Escherichia coli lacking RNAase III and containing thermolabile RNAase E and RNAase P was labeled with ³²P_i at a non-permissive temperature. RNA molecules were separated by two-dimensional polyacrylamide gel electrophoresis. Most of the small RNA species were isolated and analyzed for the presence of 5′ nucleoside triphosphates. In 16 of the 22 species analyzed a significant number of the individual molecules contained 5′ di or triphosphates. We conclude, therefore, that very little endonucleolytic RNA processing occurs in the absence of the three RNA processing enzymes RNAase III, RNAase E and RNAase P.     

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.     

Control of mRNA translation in oocytes and developing embryos of giant moths. I. Function of the 5' terminal "Cap"in the tobacco hornworm, Manduca sexta

Injection of labeled leucine into oocytes and developing embryos of the tobacco hornworm, Manduca sexta, revealed that the rate of protein synthesis increases dramatically after fertilization and continues to rise until gastrulation. Cell-free preparations of oocytes and developing embryos show a similar pattern of in vitro incorporation. When messenger RNA extracted from unfertilized oocytes was examined by gradient density centrifugation under denaturing conditions, a broad peak was observed which centered around 15 S. In contrast to mRNA extracted from oocytes, that from embryos was found to be capped by 7-methylguanosine at the 5′ terminus. When translation of oocyte mRNA was compared with that of embryo mRNA in a cell-free translation system derived from wheat germ, oocyte RNA translated less efficiently. In the presence of an inhibitor of methylation, S-adenosylhomocysteine, the differences were further widened. In competition with a cap analog, 7-methylguanosine 5′-monophosphate, embryo mRNA translation was inhibited more than oocyte at low concentrations of analog. These results are taken to indicate that the lack of a cap at the 5′ terminus could be one mechanism to inhibit translation prior to fertilization.     

RNA sequence determination in the nanogram range by a combination of in vitro labeling procedures.

Recently developed methods which allow one to read RNA sequences directly from polyacrylamide gels do not always provide unequivocal results. A combination of primary and secondary in vitro 5′-labeling, as presented here, is methodically and in its results equivalent to fingerprinting and sequencing techniques developed for in vivo labeled RNA. 5 S RNA was used to demonstrate the applicability and reliability of this combination of postlabeling procedures: 5 μg RNA was partially digested, and the resulting overlapping fragments were 5′-³²P-labeled with T4 phage-induced polynucleotide kinase in vitro. After two-dimensional polyacrylamide gel electrophoresis and carrier-free electrophoretic elution, the labeled long fragments, obtained in the 10-ng range, were completely degraded with RNase T₁ and RNase A, respectively. These digests were again ³²P-phosphorylated with T4 kinase and lead to fingerprints which allowed the deduction of the nucleotide sequences of the corresponding long fragments.     

Effect of 5-fluoroorotic acid administration of the 32 P base composition, DNA-RNA hybridization properties, and labeling of polyadenylate-rich RNA in the cytoplasm of rat liver cells

5-Fluoroorotic acid treatment lowered the (Guanine + Cytosine)/(Adenine + Uracil) base ratio of ³²P-labeled microsomal RNA from a control value of 1.36 to 1.00. Low doses of actinomycin D, which are effective in inhibiting ribosomal RNA synthesis without significantly affecting messenger RNA synthesis, caused a similar decrease in the base ratio. Microsomal RNA labeled by [³H]orotate in the presence of 5-fluoroorotic acid had approximately $\text{1}\text{2}$ the specific radioactivity but twice the hybridization efficiency of RNA labeled in its absence. Evidence is presented that this RNA (1) has a different structure from that of ribosomal RNA, (2) hybridizes to DNA with an efficiency consistent with that of other published studies of polysome-associated messenger RNA, and (3) possesses sequences which are present in other samples of liver microsomal RNA but not in kidney microsomal RNA. These properties differ from those known to be exhibited by 18 S and 28 S ribosomal RNA. Electrophoretic analysis of this [³H]orotate-labeled microsomal RNA indicated that the analogue greatly inhibited precursor incorporation into ribosomal RNA but had little or no effect on incorporation into messenger RNA. Ribosomal RNA and polyadenylate-rich nonribosomal RNA were prepared from total polyribosomes by phenol extraction at pH 7.6 and pH 9.0, respectively. 5-Fluoroorotic acid inhibited [³H]orotate or ³²P_i incorporation into the pH 7.6 fraction much more effectively than incorporation into the pH 9.0 fraction. A subfraction of the pH 9.0 RNA which was retained by a polythymidylate-cellulose column had a greatly increased adenylate content.     

The blocked and methylated 5′ terminal sequence of a specific cellular messenger: the mRNA for silk fibroin of bombyx mori

The messenger RNA for silk fibroin, labeled with ³²PO₄ and methyl-³H L-methionine, was purified to near homogeneity from the posterior silk gland of the silkworm Bombyx mori, and the sequence of a methylated, RNAase T2-resistant structure was determined. This sequence is similar structurally to 5′ terminal blocked and methylated sequences found on the total populations of polyadenylated eucaryotic cellular and certain viral mRNAs. The RNAase T2-resistant oligomer from fibroin mRNA was cleaved by nuclease P1 into three components: a blocked and methylated sequence containing three phosphates; a 2′-0-methyl UMP residue (pU^m), and an unmethylated CMP (pC). The blocked and methylated sequence comigrated in three chromatographic systems with the blocked and methylated terminus of silkworm cytoplasmic polyhedrosis virus mRNA, which has the structure m⁷GpppA^m. The fibroin mRNA cap was cleaved by nucleotide pyrophosphatase to yield 7-methyl GMP (pm⁷G) and 2′-0-methyl AMP (pA^m). This sequence also appeared to be terminally located, with the m⁷G joined by a 5′-5′ pyrophosphate linkage to the A^m. It was concluded that the 5′ terminal sequence of fibroin mRNA molecules is m⁷G(5′)ppp(5′)A^mpU^mpCp. The regulation of expression of the highly specialized gene for fibroin is discussed in light of this finding.     

The addition of 5′ cap structures occurs early in hnRNA synthesis and prematurely terminated molecules are capped

After cells were labeled by brief exposure to ³H-methyl-L-methionine, the majority of labeled 5′ terminal cap I (m⁷GpppN₁mpN₂p) oligonucleotide structures were in nuclear RNA (hnRNA) molecules ～750 nucleotides or less in length. After longer label times, the proportion of cap I structures in nuclear molecules longer than mRNA rose to approximately 60% of the total, but approximately 40% of the cap I structures were still in molecules shorter than ～750 nucleotides. The cap I structures in both long and short hnRNA chains contained all four 2′ methylated nucleotides in the N₁ position in about the same proportion as in mRNA. None of the large hnRNA molecules could be demonstrated to contain 5′ pppXp termini; the only such terminus in high molecular weight RNA was pppAp which was decreased markedly by low doses of actinomycin and is presumably the terminus of pre-rRNA. These results raise the possibilities that hnRNA chains can initiate with any of the four nucleotides, that capping occurs very close to or at the start of hnRNA chain synthesis and that approximately 40% of the hnRNA chains may be prematurely terminated.     

Studies of the Rous sarcoma virus RNA: characterization of the 5'-terminus

The 5′ terminus of the Rous Sarcoma Viral 30-40S RNA was characterized as follows: Unlabeled RNA was treated with polynucleotide kinase and (γ-³²P) ATP. Degradation of the 5′-(³²P) RNA with alkali yielded labeled pAp while degradation with venom phosphodiesterase yielded labeled 5′-AMP. Dephosphorylation with alkaline phosphatase was unnecessary for the RNA to accept³²P indicating the presence of 5′-OH ends. This establishes that the base at the 5′ end of Rous Sarcoma Viral 30-40S RNA is adenine.     

Sequence analysis of small amounts of nonradioactive oligoribonucleotides containing ribose-methylated nucleosides by a combination of 3H- and 32P-labeling techniques

The oligonucleotides A-G-A-Cm-U and Gm-A-A-Y-A-ψ were used as model compounds to demonstrate how the complete nucleotide sequence of small amounts of nonradioactive oligoribonucleotides (0.2–0.3 nmol) can be derived by a combination of ³H-labeling procedures previously published and a new method for the characterization of 2′-O-methylated nucleosides based on enzymatic ³²P labeling. The newly developed method for the identification of ribose-methylated nucleosides entails ³²P labeling by [γ-³²P]ATP/polynucleotide kinase of the 5′-terminus of a ribonuclease T₂-stable 2′-O-methylated dinucleotide derived from the polyribonucleotide, conversion of the labeled dinucleotide to the ³²P-labeled 2′-O-methylated nucleoside 5′-monophosphate, and identification of the monophosphate by its chromatographic properties on a polyethyleneimine-cellulose thin layer. The novel method is simple, fast, and sensitive and, at present, represents the only way by which ribose-methylated nucleosides can be analyzed in small amounts (0.01 nmol) of nonradioactive oligonculeotides or RNA.