Extent of Double Strandedness in Avian Myeloblastosis Virus RNA

The extent of double strandedness of avian myeloblastosis virus 70S RNA has been determined from fluorescence measurements of the intercalation of ethidium bromide. We have shown that 50% of the nucleotides of 70S RNA in solution are in a stable helical configuration. This value does not include small helical regions that are too unstable to permit intercalation of the dye. The avian myeloblastosis virus RNA as it exists within the virion has the same degree of helicity as the free 70S RNA. Heating the free 70S RNA to 55 or 70 C, followed by cooling, does not measurably change the degree of helicity; the subunits therefore have as much helicity as the parent molecule.     

Characterization and Complexity of Wheat Developing Endosperm mRNAs

Free and membrane-bound (MB) polysomes and the corresponding polyadenylated RNAs (polyA⁺ RNAs) have been isolated from developing wheat endosperm (Triticum aestivum L.) Free and MB poly(A)⁺ RNAs, analyzed on isokinetic sucrose gradient with [³H]polyuridylic acid [poly(U)] hybridization detection, appear to be 11S to 12S in size with a 7% poly(A) tail for MB RNAs. cDNAs synthesized using both of these mRNA populations in presence of a potent RNase inhibitor (RNasin), have been used for hybridization kinetics experiments. The mean square fitting analysis of the hybridization kinetics between MB cDNA and its template reveals the presence of two abundance classes representing roughly ⅔ and ⅓ of the MB poly(A)⁺ RNAs and containing the information for approximately 75 superabundant species (21,000 copies per cell) and 750 intermediate species (530 copies per cell), respectively. The mRNA population extracted from free polysomes is divided into three abundance classes. The first one is composed of superabundant sequences which would correspond to the MB superabundant mRNAs. The free mRNAs consist of about 11,000 diverse sequences, most of them being rare sequences. Heterologous hybridizations of MB cDNAs to free mRNAs have shown that some mRNAs are common to both populations. This could be explained either by a partial contamination or by free polysomes en route to their membrane destination. Contrary to the low number of diverse mRNAs corresponding to the legume seed storage proteins, the wheat endosperm superabundant mRNAs consist of about 75 different sequences which would encode most of the seed storage proteins, especially gliadins.     

A small, unstable RNA molecule of Escherichia coli: spot 42 RNA. II. Accumulation and distribution.

We have studied the accumulation and distribution of a small, unstable RNA molecule of Escherichia coli called spot 42 RNA (Ikemura &; Dahlberg, 1973b). This molecule has sequence and structural features characteristic of messenger RNAs and leader RNAs (Sahagan &; Dahlberg, 1979). It is present in 100 to 200 copies per cell when cells are grown in media supplemented with glucose. Although it accumulates in cells that have been deprived of an essential amino acid or phosphate or ammonium ions, the amount of spot 42 RNA is greatly reduced when cells are grown on non-glucose carbon sources or when cyclic AMP is added to a glucose-grown culture. Thus accumulation of spot 42 RNA is inversely proportional to the intracellular concentration of cAMP.About half of the spot 42 RNA in a cell is isolated in association with bacterial nucleoids. It is the only 4 to 6 S RNA which is enriched in this fraction. This molecule also cofractionates with 5 S ribosomal RNA during the preparation of ribosomes and ribosome fractions.The possibility that spot 42 RNA is a leader sequence whose level of accumulation is modulated by the intracellular level of cAMP is discussed.     

Properties of visna virus particles harvested at short time intervals: RNA content, infectivity, and ultrastructure

The major RNA component of Visna virus harvested at short intervals of time (5 min) is not the 60 to 70S RNA but a molecule of higher electrophoretic mobility. This RNA has been isolated and characterized. Its sedimentation coefficient is identical to that of 30 to 40S RNA subunits obtained by heat denaturation of the 60 to 70S RNA. In 1.8% acrylamide gels without agarose the electrophoretic mobility of 30 to 40S RNA subunits present in rapidly harvested virus is slightly lower than that of the subunits obtained by denaturation of the 60 to 70S RNA; after heat denaturation the mobilities are identical. These free RNA subunits present in early virus particles assemble into a 60 to 70S RNA complex as shown by following the RNA content of early virus incubated at 37 C for various lengths of time. The rate of this maturation process is slow. There is no difference between the infectivity of immature and mature virus particles. Both particles have a dense core when examined in sections of virus pellets.     

Semliki Forest Virus-Specific RNAs Synthesized In Vitro by Enzyme from Infected BHK Cells

When Semliki Forest virus (SFV)-infected BHK cells were disrupted 4 h after infection, 75 to 90% of the total virus-specific RNA synthesizing enzyme was found in the large particle fraction, along with 75 to 90% of the in vivo-synthesized double-stranded RNAs. The RNA products of this enzyme-template complex in an in vitro system were double-stranded RNAs sedimenting predominantly at 18S, and single-stranded RNAs sedimenting at 42S, 26S, and 22S. The various virus-specific SFV RNAs synthesized in vitro were associated with different sized structures, and thus each was separable by differential centrifugation. Kinetic and pulse-chase experiments showed that the double-stranded RNAs were the precursors to the single-stranded RNAs. There were several double-stranded RNAs identified both in the in vitro product and also in extracts from infected cells. The major replicative form had a molecular weight of 4.4 × 10⁶.     

Characterization of Late Polyoma mRNA

Polyoma-infected mouse kidney cell cultures were labeled with [³H]uridine for 3 h late in the lytic cycle (26 to 29 h after infection) and RNA was extracted from cytoplasm and nuclei and from isolated polyribosomes. Sedimentation velocity analysis in sucrose gradients showed that polyoma-specific “giant” and 26S RNAs are present exclusively in the nucleus. RNA associated with cytoplasmic polyribosomes was analyzed by sedimentation in aqueous sucrose density gradients and dimethylsulfoxide sucrose gradients, as well as by polyacrylamide gel electrophoresis. Polyoma-specific RNA in polyribosomes consists of at least two classes, with sedimentation coefficients of 16 (major fraction) and 19S (minor fraction) in aqueous sucrose gradients and 15 and 17S, respectively, in dimethylsulfoxide gradients. Estimates based on dimethylsulfoxide gradient and analysis suggest a molecular weight of approximately 500,000 for 16S RNA and 700,000 for 19S RNA. These polyoma RNAs seem to undergo reversible conformational changes under the different conditions of analysis. We cannot exclude the possibility that they contain more than one molecular species.     

The Fascinating World of RNA Interference

Micro- and short-interfering RNAs represent small RNA family that are recognized as critical regulatory species across the eukaryotes. Recent high-throughput sequencing have revealed two more hidden players of the cellular small RNA pool. Reported in mammals and Caenorhabditis elegans respectively, these new small RNAs are named piwi-interacting RNAs (piRNAs) and 21U-RNAs. Moreover, small RNAs including miRNAs have been identified in unicellular alga Chlamydomonas reinhardtii, redefining the earlier concept of multi-cellularity restricted presence of these molecules. The discovery of these species of small RNAs has allowed us to understand better the usage of genome and the number of genes present but also have complicated the situation in terms of biochemical attributes and functional genesis of these molecules. Nonetheless, these new pools of knowledge have opened up avenues for unraveling the finer details of the small RNA mediated pathways.     

The small nuclear RNAs of the cellular slime mold Dictyostelium discoideum. Isolation and characterization

Three species of small nuclear RNA from the lower eucaryote Dictyostelium discoideum have been isolated and characterized with regard to size, cellular abundance, modified nucleotide content, and 5'-end structures. Previous studies had shown that the nuclei of mammalian cells contain a number of discrete low molecular weight, nonribosomal, nontransfer RNA molecules known as small nuclear RNAs. The mammalian small nuclear RNAs range in size from approximately 100 to 250 nucleotides and are quite abundant, in some cases approaching ribosomal RNA in number of copies/cell. Some of these molecules have an unusual cap structure at their 5'-ends similar to that found on eucaryotic messenger RNAs, and a number contain a characteristic set of internal modifications as well. Our results indicate that the small nuclear RNAs of Dictyostelium resemble their counterparts in higher eucaryotic cells structurally, but are present in significantly fewer copies/cell. The implications of these findings for small nuclear RNA function are discussed.     

Evolutionary History of 4.5SI RNA and Indication That It Is Functional

To date, the small nuclear 4.5S_I RNA has only been studied in the rat (Rattus norvegicus). Combining PCR and hybridization analyses, we have revealed 4.5S_I RNA homologues sequences in the genomes of four myomorph rodent families (Muridae, Cricetidae, Spalicidae, and Rhizomyidae), and not in other myomorph families (Dipodidae, Zapodidae, Geomyidae, and Heteromyidae) or sciuromorph and caviomorph rodents. By Northern-hybridization, 4.5S_I RNA has been detected in the common rat (R. norvegicus, Muridae), golden hamster (Mesocricetus auratus, Cricetidae), and Russian mole rat (Spalax microphthalmus, Spalacidae), but not in the related great jerboa (Allactaga jaculus, Dipodidae) or in four non-myomorph rodent species tested. cDNA derived from 4.5S_I RNA of M. auratus and S. microphthalmus has been cloned and sequenced. The hamster RNA is found to differ from rat 4.5S_I RNA by only one nucleotide substitution. For the mole rat, two variants of 4.5S_I RNA are detected: short (S) and long (L) with length 101 and 108 nt, respectively. The L variant differs from the S variant as well as from murid and cricetid 4.5S_I RNAs by both a 7 nt insertion and a varying number of nucleotide substitutions. The sequence similarity between the spalacid S-variant and murid/crecitid variants of 4.5S_I RNA is 90%. Judging from species distribution, 4.5S_I RNA genes emerged during the same period of time as the related short interspersed element B2 arose. This occurred after the divergence of Dipodidae lineage but before the branching of Spalicidae/Rhizomyidae lineage from a common myomorph rodent stem. S variant genes seemed to emerge in a common ancestor of spalacids and rhizomyds whereas L variant genes formed in spalacids following the divergence of these two families. The low rate of evolutionary changes of 4.5S_I RNA, at least, in murids and cricetids (6 × 10⁻⁴ substitutions per site per million years), suggests that this RNA is under selection constraint and have a function. This is a remarkable fact if the recent origin and narrow species distribution range of 4.5S_I RNA genes is taken into account. Genes with narrow species distribution are proposed to be referred to as stenogenes. Received: 11 December 2000 / Accepted: 27 August 2001     

Properties of a discrete high molecular weight poly(A) containing mitochondrial RNA

Pulse-labeled mitochondrial RNA from hamster cells contains a number of discrete high molecular weight poly[A(+)] and poly[A(?)] RNAs. Characterization of the largest and most plentiful of the poly[A(+)] RNAs, the “20S_E RNA,” showed it to be a labile, unmethylated component with a molecular weight ～- 730,000. Hybridization of the 20S_E RNA to mtDNA was 60–70% inhibited in the presence of excess 17S rRNA, suggesting a significant degree of primary sequence homology between these RNA species. In vitro treatment with RNAse III converted the 20S_E RNA to a poly[A(?)] “17S” product, while similar treatment of mitochondrial 17S rRNA or a poly[A(+)] 12S_E RNA had no effect on these RNAs. These data support the proposition that the 20S_E RNA is a precursor to the 17S rRNA.     

Biosynthesis of transfer RNA: isolation and characterization of precursors to transfer RNA in the posterior silkgland of Bombyx mori.

Distinct low molecular weight RNA species that have properties expected for the precursor to tRNA have been isolated from the posterior silkglands of the silkworm Bombyx mori. These RNAs migrate between 4 S and 5 S markers on acrylamide gels and are labeled preferentially in vivo in relation to tRNA. The precursor RNAs can be converted specifically into molecules indistinguishable in size from tRNA upon incubation with “cleavage” enzymes isolated from the silkgland ribosomes. Two of the three low molecular weight RNAs contain the modified residues, pseudouridine, dihydrouridine and ribothymidine, and are methylated in vivo, suggesting that these base modifications occur while the tRNA is still in its precursor stage.     

Cloning and Expression Studies of Novel Small RNAs in Tetraploid Cotton

Small RNAs are a group of non-coding RNAs that downregulate gene expression in a sequence-specific manner to control plant growth and development. The objective of the present study was to clone and characterize several small RNAs in cotton. To identify small RNAs that are involved in the development of cotton bolls and fibers, we generated cDNA libraries from cotton bolls at 13?days post-anthesis from two cotton cultivars, Pima Phy 76 (Gossypium bardadense) and Acala 1517?C99 (Gossypium hirsutum). Screening of these libraries identified eight small RNAs, seven of which have not been reported in other plant species and appear to be absent in the known sequences of other plant species. Their predicted target genes are known to be involved in cotton fiber development. The cloned small RNAs displayed lower and differential expression in the examined boll developmental stages using RT-PCR and quantitative RT-PCR. The genetic polymorphism of the small RNAs at the DNA level was evaluated by miRNA-amplified fragment length polymorphism (AFLP) analysis using primers designed from the small miRNA genes in combination with AFLP primers. Homologous small RNA gene sequences were further isolated using this homology-based genotyping approach, and potential hairpin structures were identified. The results represent a novel method to isolate small including miRNA genes at the RNA and DNA levels in many plant species where genome sequences are not available or expressed sequence tags are limited.     

Behavior of individual maternal pA+ RNAs during embryogenesis of Xenopus laevis

Of the 10 Xenopus oocyte cDNA clones previously examined in this laboratory (L. Golden, U. Schafer, and M. Rosbash, 1980, Cell22, 835–844), 5 are complementary to RNAs which which decrease in abundance during early development. We have further examined the behavior during embryogenesis of these 5 sets of clone-complementary RNAs. The results indicate that for 3 of these 5 sets of RNAs there is an increase in the per embryo levels of RNA. Thus, 8 of the 10 clones originally examined are complementary to RNAs which increase in amount during early embryogenesis. One of the remaining two clones is complementary to (at least) 4 RNAs which vary somewhat in their levels during embryogenesis. The last clone (XOC 2–7) is complementary to an RNA species which is largely destroyed at late blastula or early gastrula. This RNA is therefore the only maternal sequence, of the ten clones examined, which unambiguously decreases in amount during embryogenesis. The data also show that XOC 2–7 RNA is largely adenylated at oocyte maturation and then deadenylated during subsequent embryogenesis while another clone, XOC 1–2, is largely dead-enylated at oocyte maturation. The results also suggest that a large fraction of oocyte RNAs are present in early embryos (and in liver) and are largely the same size as in oocytes.     

siRNAs from miRNA sites mediate DNA methylation of target genes

Arabidopsis microRNA (miRNA) genes (MIR) give rise to 20- to 22-nt miRNAs that are generated predominantly by the type III endoribonuclease Dicer-like 1 (DCL1) but do not require any RNA-dependent RNA Polymerases (RDRs) or RNA Polymerase IV (Pol IV). Here, we identify a novel class of non-conserved MIR genes that give rise to two small RNA species, a 20- to 22-nt species and a 23- to 27-nt species, at the same site. Genetic analysis using small RNA pathway mutants reveals that the 20- to 22-nt small RNAs are typical miRNAs generated by DCL1 and are associated with Argonaute 1 (AGO1). In contrast, the accumulation of the 23- to 27-nt small RNAs from the miRNA-generating sites is dependent on DCL3, RDR2 and Pol IV, components of the typical heterochromatic small interfering RNA (hc-siRNA) pathway. We further demonstrate that these MIR-derived siRNAs associate with AGO4 and direct DNA methylation at some of their target loci in trans. In addition, we find that at the miRNA-generating sites, some conserved canonical MIR genes also produce siRNAs, which also induce DNA methylation at some of their target sites. Our systematic examination of published small RNA deep sequencing datasets of rice and moss suggests that this type of dual functional MIRs exist broadly in plants.