Does Q beta replicase synthesize RNA in the absence of template?

Q beta replicase, in the absence of added template, will synthesize RNA autocatalytically. A variety of small RNa species, termed '6S RNAs' are generated. As this reaction purportedly occurs in the absence of template, it has been termed 'de novo' RNA synthesis. The question of whether Q beta replicase can polymerize replicatable RNA molecules, without instruction from a template, has important evolutionary implications. The finding that Q beta replicase was able to synthesize RNA de novo was based on (1) failure to find contaminating RNA in Q beta replicase preparations; (2) differences in the sizes of products of apparently identical reactions; and (3) kinetic differences between template-instructed and de novo reactions. Here wer describe a procedure for production of Q beta replicase lacking one of its subunits, ribosomal protein S1, involving column chromatography in the presence of a low concentration of urea. We show that the resulting highly purified enzyme will not synthesize detectable RNA in the absence of added template. We show also that the ability to perform a reaction kinetically indistinguishable from the de novo synthesis reaction can be restored to the highly purified enzyme by adding a heat-stable, alkali-labile component of Q beta replicase preparations. Thus our findings suggest that, in the novo reaction, Q beta replicase is replicating previously undetected contaminating RNA molecules.     

Template-free RNA synthesis by Q beta replicase

In the absence of extraneously added template, standard preparations of Q beta replicase spontaneously synthesize RNA in vitro, possibly as a result of RNA contamination. Using special enzyme purifications, Sumper and Luce presented evidence that self-replicating RNA not present ab initio can grow out of 'template-free' incorporation mixtures. In contrast to DNA polymerase I and RNA polymerase, which also show de novo synthesis, the products synthesized 'de novo' by Q beta replicase are RNA species containing nonrepetitive sequences of defined lengths which differ between experiments, even when synthesized under identical conditions, in fingerprints, chain lengths and kinetic parameters. Kinetic analysis of the de novo processes distinguished it from template-instructed synthesis and excluded an assumption of self-replicating RNA contamination. These conclusions were questioned recently by Hill and Blumenthal, who claimed to show that highly purified Q beta replicase preparations cannot produce RNA de novo. We now present evidence that, under the conditions required for de novo synthesis, Q beta replicase prepared according to their method is also capable of de novo synthesis. Furthermore, we show that Q beta replicase condenses nucleoside triphosphates to more or less random oligonucleotides.     

Construction of cDNA library, nucleotide sequence and analysis of entire genome of classical swine fever virus strain Shimen

According to the previously published CSFV sequences, 18 pairs of primers have been designed and synthesized, which cover the entire genome of CSFV strain Shimen. Each cDNA fragment has been amplified by RT-PCR from the anticoagulant blood of strain Shimen infected pig. The PCR products have been cloned respectively and sequenced. Results show that the cDNA library of strain Shimen and its nucleotide sequence have been obtained. The genomic RNA of strain Shimen is 12 298 nucleotides in length, containing a 5' and a 3' noncoding region 373 and 231 nt long respectively. The center of genome is a single large open reading frame of 11 697 nt which encodes a polyprotein of 3 898 amino acids. The entire sequence of strain Shimen has also been compared with that of other CSFV strains.     

Construction of cDNA library, nucleotide sequence and analysis of entire genome of classical swine fever virus strain Shimen

According to the previously published CSFV sequences, 18 pairs of primers have been designed and synthesized, which cover the entire genome of CSFV strain Shimen. Each cDNA fragment has been amplified by RT-PCR from the anticoagulant blood of strain Shimen infected pig. The PCR products have been cloned respectively and sequenced. Results show that the cDNA library of strain Shimen and its nucleotide sequence have been obtained. The genomic RNA of strain Shimen is 12 298 nucleotides in length, containing a 5′ and a 3′ noncoding region 373 and 231 nt long respectively. The center of genome is a single large open reading frame of 11 697 nt which encodes a polyprotein of 3 898 amino acids. The entire sequence of strain Shimen has also been compared with that of other CSFV strains.     

Synthesis of brome mosaic virus subgenomic RNA in vitro by internal initiation on (-)-sense genomic RNA

The genomes of many (+)-stranded RNA viruses, including plant viruses and alphaviruses, consist of polycistronic RNAs whose internal genes are expressed via subgenomic messenger RNAs. The mechanism(s) by which these subgenomic mRNAs arise are poorly understood. Based on indirect evidence, three models have been proposed: (1) internal initiation by the replicase on the (-)-strand of genomic RNA, (2) premature termination during (-)-strand synthesis, followed by independent replication of the subgenomic RNA and (3) processing by nuclease cleavage of genome-length RNA. Using an RNA-dependent RNA polymerase (replicase) preparation from barley leaves infected with brome mosaic virus (BMV) to synthesize the viral subgenomic RNA in vitro, we now provide evidence that subgenomic RNA arises by internal initiation on the (-)-strand of genomic RNA. We believe that this also represents the first in vitro demonstration of a replicase from a eukaryotic (+)-stranded RNA virus capable of initiating synthesis of (+)-sense RNA.     

lncRNAs transactivate STAU1-mediated mRNA decay by duplexing with 3' UTRs via Alu elements

Staufen 1 (STAU1)-mediated messenger RNA decay (SMD) involves the degradation of translationally active mRNAs whose 3'-untranslated regions (3' UTRs) bind to STAU1, a protein that binds to double-stranded RNA. Earlier studies defined the STAU1-binding site within ADP-ribosylation factor 1 (ARF1) mRNA as a 19-base-pair stem with a 100-nucleotide apex. However, we were unable to identify comparable structures in the 3' UTRs of other targets of SMD. Here we show that STAU1-binding sites can be formed by imperfect base-pairing between an Alu element in the 3' UTR of an SMD target and another Alu element in a cytoplasmic, polyadenylated long non-coding RNA (lncRNA). An individual lncRNA can downregulate a subset of SMD targets, and distinct lncRNAs can downregulate the same SMD target. These are previously unappreciated functions of non-coding RNAs and Alu elements. Not all mRNAs that contain an Alu element in the 3' UTR are targeted for SMD even in the presence of a complementary lncRNA that targets other mRNAs for SMD. Most known trans-acting RNA effectors consist of fewer than 200 nucleotides, and these include small nucleolar RNAs and microRNAs. Our finding that the binding of STAU1 to mRNAs can be transactivated by lncRNAs uncovers an unexpected strategy that cells use to recruit proteins to mRNAs and mediate the decay of these mRNAs. We name these lncRNAs half-STAU1-binding site RNAs (1/2-sbsRNAs).     

Complete nucleotide sequence of bacteriophage MS2 RNA: primary and secondary structure of the replicase gene.

Bacteriophage MS2 RNA is 3,569 nucleotides long. The nucleotide sequence has been established for the third and last gene, which codes for the replicase protein. A secondary structure model has also been proposed. Biological properties, such as ribosome binding and codon interactions can now be discussed on a molecular basis. As the sequences for the other regions of this RNA have been published already, the complete, primary chemical structure of a viral genome has now been established.     

The pathogenicity on legumes of Cucumber mosaic virus was determined by 243 nucleotides on 2a polymerase gene of viral RNA2

We had isolated and identified two Cucumber mosaic virus (CMV) isolates, the CMV red bean (CMV-RB)isolate and the CMV pea (CMV-P1) isolate. CMV-RBinduces necrotic local lesions on inoculated leaves of broad bean, pea, cowpea and bean, and could not infect these hosts systemically. However, CMV-P1 was able to infect these legumes systemically. To study the difference of pathogenicity. on the legumes induced by these two CMV isolates, the full-length infectious cDNA clones of CMV-Fny, which induced similar symptoms as CMV-RB in the four legumes,were used. The 243 nucleotides fragment, which encodes highly conserved GDD amino acid motif on 2a replicase gene of CMV-Fny RNA2, was replaced with that of CMV-P1. The constructed chimeric virus FP could infect these legumes systemically. The exchange of this region changes the virus symptoms on the legumes, indicating that this 243 nucleotides fragment has major effect on pathogenicity of CMV on the legumes.``     

Internal sequences that distinguish yeast from metazoan U2 snRNA are unnecessary for pre-mRNA splicing

U2 small nuclear RNA is a highly conserved component of the eukaryotic cell nucleus involved in splicing messenger RNA precursors. In the yeast Saccharomyces cerevisiae, U2 RNA interacts with the intron by RNA-RNA pairing between the conserved branchpoint sequence UACUAAC and conserved nucleotides near the 5' end of U2 (ref. 4). Metazoan U2 RNA is less than 200 nucleotides in length, but yeast U2 RNA is 1,175 nucleotides long. The 5' 110 nucleotides of yeast U2 are homologous to the 5' 100 nucleotides of metazoan U2 (ref. 6), and the very 3' end of yeast U2 bears a weak structural resemblance to features near the 3' end of metazoan U2. Internal sequences of yeast U2 share primary sequence homology with metazoan U4, U5 and U6 small nuclear RNA (ref. 6), and have regions of complementarity with yeast U1 (ref. 7). We have investigated the importance of the internal U2 sequences by their deletion. Yeast cells carrying a U2 allele lacking 958 nucleotides of internal U2 sequence produce a U2 small nuclear RNA similar in size to that found in other organisms. Cells carrying only the U2 deletion grow normally, have normal levels of spliced mRNA and do not accumulate unspliced precursor mRNA. We conclude that the internal sequences of yeast U2 carry no essential function. The extra RNA may have a non-essential function in efficient ribonucleoprotein assembly or RNA stability. Variation in amount of RNA in homologous structural RNAs has precedence in ribosomal RNA and RNaseP.     

The pathogenicity on legumes of Cucumber mosaic virus was determined by 243 nucleotides on 2a polymerase gene of viral RNA2

We had isolated and identified two Cucumber mosaic virus (CMV) isolates, the CMV red bean (CMV-RB) isolate and the CMV pea (CMV-P1) isolate. CMV-RB induces necrotic local lesions on inoculated leaves of broad bean, pea, cowpea and bean, and could not infect these hosts systemically. However, CMV-P1 was able to infect these legumes systemically. To study the difference of pathogenicity on the legumes induced by these two CMV isolates, the full-length infectious cDNA clones of CMV-Fny, which induced similar symptoms as CMV-RB in the four legumes, were used. The 243 nucleotides fragment, which encodes highly conserved GDD amino acid motif on 2a replicase gene of CMV-Fny RNA2, was replaced with that of CMV-P1. The constructed chimeric virus FP could infect these legumes systemically. The exchange of this region changes the virus symptoms on the legumes, indicating that this 243 nucleotides fragment has major effect on pathogenicity of CMV on the legumes.     

Prevalence of off-target effects in Drosophila RNA interference screens

RNA interference (RNAi) in both plants and animals is mediated by small RNAs of approximately 21-23 nucleotides in length for regulation of target gene expression at multiple levels through partial sequence complementarities. Combined with widespread genome sequencing, experimental use of RNAi has the potential to interrogate systematically all genes in a given organism with respect to a particular function. However, owing to a tolerance for mismatches and gaps in base-pairing with targets, small RNAs could have up to hundreds of potential target sequences in a genome, and some small RNAs in mammalian systems have been shown to affect the levels of many messenger RNAs besides their intended targets. The use of long double-stranded RNAs (dsRNAs) in Drosophila, where Dicer-mediated processing produces small RNAs inside cells, has been thought to reduce the probability of such 'off-target effects' (OTEs). Here we show, however, that OTEs mediated by short homology stretches within long dsRNAs are prevalent in Drosophila. We have performed a genome-wide RNAi screen for novel components of Wingless (Wg) signal transduction in Drosophila S2R + cells, and found few, if any, legitimate candidates. Rather, many of the top candidates exert their effects on Wg response through OTEs on known pathway components or through promiscuous OTEs produced by tandem trinucleotide repeats present in many dsRNAs and genes. Genes containing such repeats are over-represented in candidate lists from published screens, suggesting that they represent a common class of false positives. Our results suggest simple measures to improve the reliability of genome-wide RNAi screens in Drosophila and other organisms.     

Conservation of the sequence and temporal expression of let-7 heterochronic regulatory RNA

Two small RNAs regulate the timing of Caenorhabditis elegans development. Transition from the first to the second larval stage fates requires the 22-nucleotide lin-4 RNA, and transition from late larval to adult cell fates requires the 21-nucleotide let-7 RNA. The lin-4 and let-7 RNA genes are not homologous to each other, but are each complementary to sequences in the 3' untranslated regions of a set of protein-coding target genes that are normally negatively regulated by the RNAs. Here we have detected let-7 RNAs of approximately 21 nucleotides in samples from a wide range of animal species, including vertebrate, ascidian, hemichordate, mollusc, annelid and arthropod, but not in RNAs from several cnidarian and poriferan species, Saccharomyces cerevisiae, Escherichia coli or Arabidopsis. We did not detect lin-4 RNA in these species. We found that let-7 temporal regulation is also conserved: let-7 RNA expression is first detected at late larval stages in C. elegans and Drosophila, at 48 hours after fertilization in zebrafish, and in adult stages of annelids and molluscs. The let-7 regulatory RNA may control late temporal transitions during development across animal phylogeny.     

Control of RNase E-mediated RNA degradation by 5'-terminal base pairing in E. coli.

Despite the variety of messenger RNA half-lives in bacteria (0.5-30 min in Escherichia coli) and their importance in controlling gene expression, their molecular basis remains obscure. The lifetime of an entire mRNA molecule can be determined by features near its 5' end, but no 5' exoribonuclease has been identified in any prokaryotic organism. A mutation that inactivates E. coli RNase E also increases the average lifetime of bulk E. coli mRNA and of many individual messages, suggesting that cleavage by this endonuclease may be the rate-determining step in the degradation of most mRNAs in E. coli. We have investigated the substrate preference of RNase E in E. coli by using variants of RNA I, a small untranslated RNA whose swift degradation in vivo is initiated by RNase E cleavage at an internal site. We report here that RNase E has an unprecedented substrate specificity for an endoribonuclease, as it preferentially cleaves RNAs that have several unpaired nucleotides at the 5' end. The sensitivity of RNase E to 5'-terminal base pairing may explain how determinants near the 5' end can control rates of mRNA decay in bacteria.     

An endoribonuclease-prepared siRNA screen in human cells identifies genes essential for cell division

RNA interference (RNAi) is an evolutionarily conserved defence mechanism whereby genes are specifically silenced through degradation of messenger RNAs; this process is mediated by homologous double-stranded (ds)RNA molecules. In invertebrates, long dsRNAs have been used for genome-wide screens and have provided insights into gene functions. Because long dsRNA triggers a nonspecific interferon response in many vertebrates, short interfering (si)RNA or short hairpin (sh)RNAs must be used for these organisms to ensure specific gene silencing. Here we report the generation of a genome-scale library of endoribonuclease-prepared short interfering (esi)RNAs from a sequence-verified complementary DNA collection representing 15,497 human genes. We used 5,305 esiRNAs from this library to screen for genes required for cell division in HeLa cells. Using a primary high-throughput cell viability screen followed by a secondary high content videomicroscopy assay, we identified 37 genes required for cell division. These include several splicing factors for which knockdown generates mitotic spindle defects. In addition, a putative nuclear-export terminator was found to speed up cell proliferation and mitotic progression after knockdown. Thus, our study uncovers new aspects of cell division and establishes esiRNA as a versatile approach for genomic RNAi screens in mammalian cells.