Interaction of viral RNA with Escherichia coli. 3. Synthesis of poliovirus-specific RNA directed by isolated poliovirus RNA

Poliovirus RNA directs the synthesis of virus-specific RNA in E. coli as reported previously for poliovirus-induced double-stranded RNA. Synthesis of viral RNA can be followed by conversion of viral RNA into a double-stranded RNase-resistant state, by increase in infectivity and by hybridization of newly synthesized RNA to viral RNA. Virus-specific RNA synthesis occurs also in the presence of inhibitors of protein synthesis indicating that an enzyme is present in E. coli which can use RNA as a template.     

Effects of Potassium Nutrition on Ribonucleic Acid and Ribonuclease in Zea mays L

Potassium deficiency decreased the total RNA per shoot in corn (Zea mays L.) seedlings due to the reduced sizes of the plant, but increased the ratio of RNA to dry matter as much as 40%. Total base composition of RNA was unaffected by K⁺ deficiency. Thus K⁺ deficiency does not appear to alter RNA metabolism to such an extent that a lack of RNA becomes a factor limiting growth. The RNA contents of leaves were followed as deficiency developed or as the plant recovered from deficiency. Such time-course studies, although extremely useful in microbial work, did not yield as much information with higher plants. The ratio of RNA to dry matter in younger leaves was much higher than that in older leaves. The ribonuclease level in corn shoots was increased as much as threefold by K⁺ deficiency.     

Isolation and Purification of Double-Stranded RNA Fragments from Retrovirus RNA

Genomic or high molecular weight RNA of retroviruses consisted of 2 to 5% double-stranded RNA. Fragmentation of genomic viral RNA by limited RNase T₁ treatment before cellulose CF-11 chromatography indicated that 3 to 5% of the viral RNA fragments were eluted as double-stranded RNA. This double-strandedness was in close agreement with the more representative 2 to 3% double-strandedness as determined by RNase T₂ resistance studies performed on genomic and subunit viral RNA. The double-stranded RNA of RNase T₂ treated retrovirus RNA was purified by cellulose CF-11 chromatography.     

The Sm Complex Is Required for the Processing of Non-Coding RNAs by the Exosome

A key question in the field of RNA regulation is how some exosome substrates, such as spliceosomal snRNAs and telomerase RNA, evade degradation and are processed into stable, functional RNA molecules. Typical feature of these non-coding RNAs is presence of the Sm complex at the 3′end of the mature RNA molecule. Here, we report that in Saccharomyces cerevisiae presence of intact Sm binding site is required for the exosome-mediated processing of telomerase RNA from a polyadenylated precursor into its mature form and is essential for its function in elongating telomeres. Additionally, we demonstrate that the same pathway is involved in the maturation of snRNAs. Furthermore, the insertion of an Sm binding site into an unstable RNA that is normally completely destroyed by the exosome, leads to its partial stabilization. We also show that telomerase RNA accumulates in Schizosaccharomyces pombe exosome mutants, suggesting a conserved role for the exosome in processing and degradation of telomerase RNA. In summary, our data provide important mechanistic insight into the regulation of exosome dependent RNA processing as well as telomerase RNA biogenesis.     

The maize mitochondrial plasmid RNA b is associated with protein during synthesis but is not encapsidated

RNA b is the most abundant member of a family of autonomously replicating single- and double-stranded RNA plasmids found in maize mitochondria. The extent to which this molecule is associated with proteins was investigated by rate zonal and CsCl equilibrium density gradient centrifugation of clarified lysates of S cytoplasm maize mitochondria. A soluble complex of RNA b, responsible for synthesis of the more abundant (+) RNA b strand in mitochondrial lysates, was identified. The complex had a buoyant density of 1.49 g/cm³, indicating a substantial non-nucleic acids content. The sedimentation coefficient of the complex, however, was only slightly larger than that of deproteinized RNA b. Synthesis of RNA b as well as the larger RNA plasmid, RNA a, was resistant to heparin, suggesting that, for both RNAs, preformed complexes between an RNA template and an RNA-dependent RNA polymerase capable of elongating in vivo preinitiated RNA plasmid strands, were present in the lysate. Only a small fraction of RNA b molecules were bound in the complex; the bulk of RNA b sedimented at the same rate as the deproteinized RNA. Thus, after replication, maize mitochondrial plasmids are not associated with nucleoprotein capsids although their synthesis takes place through ribonucleoprotein replication complexes.     

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.     

Histone mRNA in eggs and embryos of Strongylocentrotus purpuratus

Histone messenger RNA is detectable in both the maternal RNA which is stored in the unfertilized sea urchin egg and in the RNA species which are synthesized $\text{de}$$\text{novo}$ after fertilization. Hybridization competition experiments show that sequences similar to pulse-labeled 912S RNA from morulae are present in total RNA from unfertilized eggs as well as that from later stages. The proportion of histone mRNA in cellular RNA increases after fertilization, reaching a maximum at the morula stage. Although these messengers are still present in hatched blastulae and gastrulae, they represent a smaller proportion of total RNA compared with earlier stages.     

Human Enterovirus Nonstructural Protein 2CATPase Functions as Both an RNA Helicase and ATP-Independent RNA Chaperone

RNA helicases and chaperones are the two major classes of RNA remodeling proteins, which function to remodel RNA structures and/or RNA-protein interactions, and are required for all aspects of RNA metabolism. Although some virus-encoded RNA helicases/chaperones have been predicted or identified, their RNA remodeling activities in vitro and functions in the viral life cycle remain largely elusive. Enteroviruses are a large group of positive-stranded RNA viruses in the Picornaviridae family, which includes numerous important human pathogens. Herein, we report that the nonstructural protein 2C^ATPase of enterovirus 71 (EV71), which is the major causative pathogen of hand-foot-and-mouth disease and has been regarded as the most important neurotropic enterovirus after poliovirus eradication, functions not only as an RNA helicase that 3′-to-5′ unwinds RNA helices in an adenosine triphosphate (ATP)-dependent manner, but also as an RNA chaperone that destabilizes helices bidirectionally and facilitates strand annealing and complex RNA structure formation independently of ATP. We also determined that the helicase activity is based on the EV71 2C^ATPase middle domain, whereas the C-terminus is indispensable for its RNA chaperoning activity. By promoting RNA template recycling, 2C^ATPase facilitated EV71 RNA synthesis in vitro; when 2C^ATPase helicase activity was impaired, EV71 RNA replication and virion production were mostly abolished in cells, indicating that 2C^ATPase-mediated RNA remodeling plays a critical role in the enteroviral life cycle. Furthermore, the RNA helicase and chaperoning activities of 2C^ATPase are also conserved in coxsackie A virus 16 (CAV16), another important enterovirus. Altogether, our findings are the first to demonstrate the RNA helicase and chaperoning activities associated with enterovirus 2C^ATPase, and our study provides both in vitro and cellular evidence for their potential roles during viral RNA replication. These findings increase our understanding of enteroviruses and the two types of RNA remodeling activities.     

Recognition of two distinct elements in the RNA substrate by the RNA-binding domain of the T. thermophilus DEAD box helicase Hera

DEAD box helicases catalyze the ATP-dependent destabilization of RNA duplexes. Whereas duplex separation is mediated by the helicase core shared by all members of the family, flanking domains often contribute to binding of the RNA substrate. The Thermus thermophilus DEAD-box helicase Hera (for “heat-resistant RNA-binding ATPase”) contains a C-terminal RNA-binding domain (RBD). We have analyzed RNA binding to the Hera RBD by a combination of mutational analyses, nuclear magnetic resonance and X-ray crystallography, and identify residues on helix α1 and the C-terminus as the main determinants for high-affinity RNA binding. A crystal structure of the RBD in complex with a single-stranded RNA resolves the RNA–protein interactions in the RBD core region around helix α1. Differences in RNA binding to the Hera RBD and to the structurally similar RBD of the Bacillus subtilis DEAD box helicase YxiN illustrate the versatility of RNA recognition motifs as RNA-binding platforms. Comparison of chemical shift perturbation patterns elicited by different RNAs, and the effect of sequence changes in the RNA on binding and unwinding show that the RBD binds a single-stranded RNA region at the core and simultaneously contacts double-stranded RNA through its C-terminal tail. The helicase core then unwinds an adjacent RNA duplex. Overall, the mode of RNA binding by Hera is consistent with a possible function as a general RNA chaperone.     

Ribonucleic acid synthesis in streptomyces antibioticus: Stable ribonucleic acid species synthesized by young and old cells

In studies of RNA synthesis by intact cells and cell-free extracts of $\text{Streptomyces antibioticus}$, it has been found that 48 hr cells (producing actinomycin) and cell-free extracts are less efficient than 12 hr cells (not producing actinomycin) and extracts in the synthesis of RNA. Analysis of the products of “in vivo” and “in vitro” RNA synthesis by sucrose gradient centrifugation reveals that both 12 and 48 hr cultures and cell-free extracts synthesize ribosomal RNA as well as RNA species of higher and lower molecular weights. However, 50–60% of the ³H-uridine labelled RNA synthesized by intact cells sediments as rRNA as compared with only 5–10% of the cell-free product. The addition of 2 × 10^?5 M actinomycin D to incubation mixtures for cell-free RNA synthesis does not significantly alter the relative amounts of the various RNA species synthesized by 12 or 48 hr extracts.     

When one is better than two: RNA with dual functions

The central dogma of biology, until not long ago, held that genetic information stored on DNA molecules was translated into the final protein products through RNA as intermediate molecules. Then, an additional level of complexity in the regulation of genome expression was added, implicating new classes of RNA molecules called non-coding RNA (ncRNA). These ncRNA are also often referred to as functional RNA in that, although they do not contain the capacity to encode proteins, do have a function as RNA molecules. They have been thus far considered as truly non-coding RNA since no ORF long enough to be considered, nor protein, have been associated with them. However, the recent identification and characterization of bifunctional RNA, i.e. RNA for which both coding capacity and activity as functional RNA have been reported, suggests that a definite categorization of some RNA molecules is far from being straightforward.Indeed, several RNA primarily classified as non-protein-coding RNA has been showed to hold coding capacities and associated peptides. Conversely, mRNA, usually regarded as strictly protein-coding, may act as functional RNA molecules. Here, we describe several examples of these bifunctional RNA that have been already characterized from bacteria to mammals. We also extend this concept to fortuitous acquisition of dual function in pathological conditions and to the recently highlighted duality between information carried by a gene and its pseudogenes counterparts.     

The naturally trans-acting ribozyme RNase P RNA has leadzyme properties

Divalent metal ions promote hydrolysis of RNA backbones generating 5′OH and 2′;3′P as cleavage products. In these reactions, the neighboring 2′OH act as the nucleophile. RNA catalyzed reactions also require divalent metal ions and a number of different metal ions function in RNA mediated cleavage of RNA. In one case, the LZV leadzyme, it was shown that this catalytic RNA requires lead for catalysis. So far, none of the naturally isolated ribozymes have been demonstrated to use lead to activate the nucleophile. Here we provide evidence that RNase P RNA, a naturally trans-acting ribozyme, has leadzyme properties. But, in contrast to LZV RNA, RNase P RNA mediated cleavage promoted by Pb²⁺ results in 5′ phosphate and 3′OH as cleavage products. Based on our findings, we infer that Pb²⁺ activates H₂O to act as the nucleophile and we identified residues both in the substrate and RNase P RNA that most likely influenced the positioning of Pb²⁺ at the cleavage site. Our data suggest that Pb²⁺ can promote cleavage of RNA by activating either an inner sphere H₂O or a neighboring 2′OH to act as nucleophile.     

Synthesis of mitochondrial RNA in disaggregated embryos of Xenopus laevis

Mitochondrial RNA synthesis was studied during early Xenopus laevis development using disaggregated embryos. By gel electrophoresis, the labeled mitochondrial RNA consisted of three discrete species. Transfer RNA was the only mitochondrial RNA species which was methylated. Incorporation of ³²P into mitochondrial RNA was detectable as early as the blastula stages. The level of incorporation was found to increase with increasing developmental age. All species of mitochondrial RNA appeared to be labeled at a constant rate relative to one another. Partial analysis of the acid-soluble nucleotide pool indicated that the observed incorporation probably represents a net increase in the synthetic rate for mitochondrial RNA as development proceeds.     

Evidence that multiple species of aminoacylated transfer RNA are present in regenerating optic axons of goldfish

This study reports that 4S RNA present in regenerating optic axons of goldfish is likely to be transfer RNA. Evidence is also presented which indicates that this transfer RNA is similar to transfer RNA found in tectal cells and that its aminocylation is likely to occur both in retinal ganglion cells prior to axonal transport as well as in the axon itself. Fish with regenerating optic nerves received intraocular injections of [³H]uridine followed 4 days later by intracranial injections of [¹⁴C]uridine. Radioactive tectal 4S RNA was isolated 6 days after [³H]uridine injections and chromatographed by BD cellulose chromatography. Optical density as well as radioactivity profiles for both [¹⁴C]4S RNA (from tectal cells) and [³H]4S RNA (90% of which originated from regenerating optic axons) were found to be similar toE. coli transfer RNA optical density profiles, indicating that the intra-axonal 4S RNA is likely to be transfer RNA. Moreover, comparisons of³H/¹⁴C suggest that intra-axonal and cellular 4S RNAs are composed of similar species of transfer RNA. Results of other experiments indicated that aminoacylation of axonally transported tRNA occurs both in the retina and in optic axons subsequent to axonal transport.