Resolution of the 1H-1H NOE spectrum of RNA into three dimensions using 15N-1H two-bond couplings

The feasibility of using two-bond ¹⁵N-¹H couplings to resolve the ¹H-¹H nuclear Overhauser effect spectrum of RNA into a third dimension was investigated, using the 36-nucleotide gene 32 messenger RNA pseudoknot of bacteriophage T2 as an example. The two-bond ¹⁵N-¹H couplings present in adenosine and guanosine were found to be suitable for generating a three-dimensional ¹H-¹H-¹⁵N NOESY-HSQC spectrum with reasonably good sensitivity, as well as favorable chemical shift dispersion in the nitrogen dimension. The described NMR experiment provides a tool that can be used to complement other heteronuclear methods in the analysis of RNA structure.     

A comparative study of three closely related cytoplasmic polyhedrosis viruses

A cytoplasmic polyhedrosis virus (CPV) from the pine caterpillar, Dendrolimus spectabilis, was compared with Japanese isolates of closely related viruses from the silkworm, Bombyx mori, and gypsy moth, Lymantria dispar. The sizes of the viral RNA genome segments were almost identical, although the CPVs from D. spectabilis and L. dispar could be distinguished from the silkworm virus by a small size difference (0.03 × 10⁶ daltons) in one segment. The same viruses were also distinguishable by RNA homology differences of 25–50% measured by the reannealing of ³H-labeled single-stranded viral messenger RNA (synthesized in vitro) to heat-denatured viral double-stranded RNA. Antigenic differences were also detected by gel immunodiffusion tests. CPVs of D. spectabilis and L. dispar were indistinguishable by these criteria.     

Small RNA species in maize tissue

The low molecular weight RNA components of maize have been analyzed after labeling callus and leaf tissue with [³H]uridine in vitro. Electrophoresis of the isolated RNA on acrylamide slab gels reveals, apart from 5S and transfer RNA, three major and about five minor RNA species with chain lengths between 140 and 280 nucleotides. These RNA molecules are labeled as rapidly as 5S, transfer RNA, and do not represent degradation products of large ribosomal RNA molecules. Furthermore, like 5S and transfer RNA, these small RNA species are stable and show no detectable turnover within forty-eight hours. Fractionation of the tissue into crude subcellular fractions indicates a preferential association of some of the small stable RNA species with the nucleus, while others appear to be located in the cytoplasm. The low molecular weight RNA spectrum from the leaf is similar to that observed in callus, with the major small RNA species equally present in both tissues.Abbreviations tRNA transfer RNA - hnRNA heterogenous nuclear RNA - mRNA messenger RNA - scRNA small cytoplasmic RNA - snRNA small nuclear RNA     

Auxin-stimulated RNA Synthesis in Oat Coleoptile Cells

Using oat coleoptile segments the following results were obtained. Ten mg/l auxin (indole-3-acetic acid) increased the incorporation of uracil-2-¹⁴C and orthophosphate-³²P into RNA fraction during a relatively short incubation period. Stimulation of ³²P incorporation due to auxin was found only in the region heavier than ribosomal RNA, probably in the messenger RNA region. The stimulation of uracil-2-¹⁴C incorporation into RNA caused by auxin was not influenced by the presence of 0.3 M mannitol which prevents osmotically the water absorption of cells. It is concluded that auxin primarily stimulates the biosynthesis of RNA, possibly messenger, in oat coleoptile cells.     

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 size of poly(A)-containing RNAs in Drosophila melanogaster embryos

The size range of poly(A)-containing RNA from Drosophila melanogaster embryos has been estimated by hybridization with ³H-labeled poly(U) and subsequent fractionation on sucrose gradients. The median size of nuclear poly(A)-containing RNA is about 30 S (6000 nucleotides), and the median size of cytoplasmic poly(A)-containing RNA is about 17 S (1800 nucleotides). The relationship of these sizes to messenger RNA needed to code for protein and to the length of DNA contained in a chromomere is discussed.Research grant support was provided by NIH (6M35558; HD-00266) and NSF (GB-30600).     

The isolation of plasmids containing dna complementary to messenger rna for variant surface glycoproteins of Trypanosoma brucei

We have isolated poly(A)⁺ RNA from four antigenic variants (117, 118, 121, 221) of one clone of Trypanosoma brucei. Translation of these poly(A)⁺ RNAs in a rabbit reticulocyte lysate gave rise to proteins that could be precipitated with antisera against homologous variant surface glycoprotein, the protein responsible for antigenic variation in trypanosomes. From the electrophoretic mobility of these in vitro products in sodium dodecyl sulphate (SDS) gels we infer that variant surface glycoproteins (VSGs) are made as pre-proteins, which require trimming to yield mature VSGs.The total translation products from the four poly(A)⁺ RNAs produced a complex set of bands on SDS gels, which only differed in the region where the variant pre-glycoproteins migrated. The only detectable variation in the messenger RNA populations of these variants is, therefore, in the messenger RNA for variant pre-glycoproteins.We have made duplex DNA copies of these poly(A)⁺ RNAs, linked the complementary DNA to plasmid pBR322 by GC tailing and cloned this recombinant DNA in Escherichia coli. Colony hybridization with complementary DNA made on poly(A)⁺ RNA showed that 7–10% of the colonies contained DNA that hybridized only with the homologous probe. Plasmid DNA was isolated from ten such colonies (two or three of each variant complementary DNA), bound to diazobenzyloxymethyl-cellulose (DBM) paper and used to select complementary messenger RNA from total poly(A)⁺ RNA by hybridization. In eight cases the RNA recovered from the filter gave variant pre-glycoprotein as the predominant product of in vitro translation.Poly(A)⁺ RNA from each of the variants only hybridized to the homologous complementary DNA in filter hybridizations. Each trypanosome variant, therefore, contains no detectable messenger RNAs for the three heterologous variant-specific glycoproteins tested. We conclude from this lack of cross-hybridization that antigenic diversity in trypanosomes, unlike antibody diversity in mammals, does not involve the linkage of a repertoire of genes for the variable N-terminal half to a single gene for the C-terminal half of the VSGs.     

Novel insights into m6A modification of coding and non-coding RNAs in tumor biology: From molecular mechanisms to therapeutic significance

Accumulating evidence has revealed that m⁶A modification, the predominant RNA modification in eukaryotes, adds a novel layer of regulation to the gene expression. Dynamic and reversible m⁶A modification implements sophisticated and crucial functions in RNA metabolism, including generation, splicing, stability, and translation in messenger RNAs (mRNAs) and non-coding RNAs (ncRNAs). Furthermore, m⁶A modification plays a determining role in producing various m⁶A-labeling RNA outcomes, thereby affecting several functional processes, including tumorigenesis and progression. Herein, we highlighted current advances in m⁶A modification and the regulatory mechanisms underlying mRNAs and ncRNAs in distinct cancer stages. Meanwhile, we also focused on the therapeutic significance of m⁶A regulators in clinical cancer treatment.     

Messenger ribonucleic acid content of Bacillus amyloliquefaciens throughout its growth cycle compared with Bacillus subtilis 168

The messenger RNA contents of Bacillus amyloliquefaciens and B. subtilis 168, grown in a 1% maltose-0.5% casein hydrolysate complex medium, were determined throughout their growth cycles by a hybridization technique. In both cases there was a level equal to about 3% of the total cellular RNA during the exponential phase. In B. subtilis this level was maintained into the stationary phase. By contrast, in B. amyloliquefaciens the proportion of messenger RNA increased after the end of exponential growth levelling off in the stationary phase at a value twice that observed in exponential growth. The total messenger RNA in each organism was resolved into two components, that involved in the formation of cell proteins and that concerned in extracellular protein production, by determining the relative rates of incorporation of l-[¹⁴C]valine into the two protein fractions. In both cases the cell protein component was the same and remained a relatively constant proportion of the total cellular material throughout the growth cycles. The exoprotein mRNA paralleled exoprotein secretion in each species, remaining at a constant low level in B. subtilis and undergoing a tenfold increase after the end of exponential growth in B. amyloliquefaciens. Applying a serial hybridization procedure to B. amyloliquefaciens, no evidence was obtained for the accumulation of a specific component of the messenger RNA in the exponential or post-exponential phase of growth, which was not detected by hybridization.     

The properties and function of rapidly-labelled nuclear RNA

Summary Nuclei were isolated from cultured cells of Acer pseudoplatanus L. previously pulse-labelled with [5-³H]uridine or [³²P]phosphate and the properties of the rapidly-labelled RNA were studied. Polyacrylamide gel electrophoresis showed ribosomal RNA precursors and processing intermediates with molecular weights of 3.4, 2.5, 1.4 and 1×10⁶ daltons, together with polydisperse RNA. The relative proportions of ribosomal RNA precursors and polydisperse RNA varied according to the length of the labelling period, but after 30 min approximately 90% of the radioactive RNA was polydisperse. The relationship between this polydisperse RNA and messenger RNA was investigated. The percentage of total nuclear RNA retained by chromatography on oligodeoxythymidylic acid-cellulose columns varied from 6% to 16% depending on the length of the labelling period. This RNA fraction, which has an adenylic acid content of approximately 45%, is assumed to represent RNA with polyadenylic acid sequences attached. A larger proportion of the nuclear polydisperse RNA lacked polyadenylic acid. Both types of polydisperse RNA were similar in size and during polyacrylamide gel electrophoresis migrated as broad peaks with an average molecular weight of approximately 10⁶ daltons. The polydisperse nuclear RNA that lacks polyadenylic acid was found to be similar in nucleotide composition to ribosomal RNA and is assumed to represent growing chains of ribosomal precursor RNA. After short labelling times the majority of the radioactivity incorporated into nuclear RNA is present in molecules of this type. This suggests that the designation of pulse-labelled polydisperse RNA as messenger RNA or precursor to messenger RNA solely on the basis of rapid labelling and size heterogeneity is unsound. The average molecular weight of the polyadenylic acid-containing messenger RNA from the cytoplasm was less than that of the corresponding nuclear RNA (6 and 9×10⁵ daltons respectively). This suggest either that the majority of the nuclear polyadenylic acid-containing RNA does not enter the cytoplasm, or if it does, that it first undergoes a reduction in size.Abbreviations rRNA ribosomal RNA - mRNA messenger - RNA poly(A), polyadenylic acid, poly(A) and poly(A) - RNA RNA with and without poly(A) sequences attached - poly(U) polyuridylic acid - oligo (dT)-cellulose cellulose with oligo deoxythymidylic acid covalently attached - C cytidylic acid - A adenylic acid - G guanylic acid - U uridylic acid     

Regulation of Virus Replication and T Cell Homeostasis by N~6-Methyladenosine

RNA modifications are abundant in eukaryotes, bacteria, and archaea. N~6-methyladenosine(m~6A), a type of RNA modification mainly found in messenger RNA(mRNA), has significant effects on the metabolism and function of m RNAs. This modification is governed by three types of proteins, namely methyltransferases as ‘‘writers' ', demethylases as ‘‘erasers' ',and specific m~6A-binding proteins(YTHDF1-3) as ‘‘readers' '. Further, it is important for the regulation of cell fate and has a critical function in many biological processes including virus replication, stem cell differentiation, and cancer development, and exerts its effect by controlling gene expression. Herein, we summarize recent advances in research on m~6A in virus replication and T cell regulation, which is a rapidly emerging field that will facilitate the development of antiviral therapies and the study of innate immunity.     

Analysis of the coding activity and stability of messenger RNA in Drosophila oocytes.

The coding activity of the messenger RNA in the ooplasm of late stage 14 (S14) oocytes of Drosophila melanogaster was analyzed by labeling the oocytes in vitro with [³⁵S]methionine and examining the labeled products by two-dimensional gel electrophoresis and fluorography. This analysis was done both with newly formed S14 oocytes from rapidly laying females and with S14 oocytes stored for about 10 days in females that were prevented from laying. Comparison of the fluorographs showed that the proteins labeled in the newly formed oocytes were also labeled in the stored oocytes. Thus, the coding activity of S14 oocyte messenger RNA appears to remain stable during prolonged storage in utero. The oocyte proteins synthesized during oogenesis and incorporated into S14 oocytes were labeled in vivo by injecting [³⁵S]methionine into newly eclosed females, and the S14 oocytes were removed 2 days later for gel electrophoresis and fluorography. Comparison of the fluorographs produced by the in vivo and in vitro labeling procedures showed that most of the oocyte proteins labeled in vivo were also labeled in vitro. The S14 oocytes, therefore, appear to contain messenger RNA for most of the oocyte proteins synthesized during oogenesis. There were also several additional proteins detected only in the fluorographs of the in vivo labeled oocytes; the most prominent of these were identified by immunoprecipitation tests as vitellogenin proteins of yolk granules, which are known to be synthesized outside the oocyte, in fat bodies. The occurrence of stable S14 oocyte messenger RNA for most of the oocyte proteins suggests that the synthesis of those proteins during oogenesis occurs in the developing oocytes, specified by a stable population of oocyte messenger RNA.