RNA-dependent RNA polymerase activity related to the 20S RNA replicon ofSaccharomyces cerevisiae
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Abstract:
Saccharomyces cerevisiae contains two double-stranded RNA (dsRNA) viruses (L-A and L-BC) and two different single-stranded (ssRNA) replicons (20S RNA and 23S RNA). Replicase (dsRNA synthesis on a ssRNA template) and transcriptase (ssRNA synthesis on a dsRNA template) activities have been described for L-A and L-BC viruses, but not for 20S or 23S RNA. We report the characterization of a new in vitro RNA replicase activity in S. cerevisiae. This activity is detected after partial purification of a particulate fraction in CsCl gradients where it migrates at the density of free protein. The activity does not require the presence of L-A or L-BC viruses or 23S RNA, and its presence or absence is correlated with the presence or absence of the 20S RNA replicon. Strains lacking both this RNA polymerase activity and 20S RNA acquire this activity when they acquire 20S RNA by cytoduction (cytoplasmic mixing). This polymerase activity converts added ssRNA to dsRNA by synthesis of the complementary strand, but has no specificity for the 3′ end or internal template sequence. Although it replicates all tested RNA templates, it has a template size requirement, being unable to replicate templates larger than 1kb. The replicase makes dsRNA from a ssRNA template, but many single-stranded products due to a terminal transferase activity are also formed. These results suggest that, in contrast to the L-A and L-BC RNA polymerases, dissociation of 20S RNA polymerase from its RNA (or perhaps some cellular factor) makes the enzyme change its specificity.Keywords:
RNA Silencing
Small nuclear RNA
RNA polymerase I
Replicon
A previously unknown coronavirus (CoV) is the aetiological agent causing severe acute respiratory syndrome (SARS), for which an effective antiviral treatment is urgently needed. To enable the rapid and biosafe identification of coronavirus replicase inhibitors, we have generated a non-cytopathic, selectable replicon RNA (based on human CoV 229E) that can be stably maintained in eukaryotic cells. Most importantly, the replicon RNA mediates reporter gene expression as a marker for coronavirus replication. We have used a replicon RNA-containing cell line to test the inhibitory effect of several compounds that are currently being assessed for SARS treatment. Amongst those, interferon-alpha displayed the strongest inhibitory activity. Our results demonstrate that coronavirus replicon cell lines provide a versatile and safe assay for the identification of coronavirus replicase inhibitors. Once this technology is adapted to SARS-CoV replicon RNAs, it will allow high throughput screening for SARS-CoV replicase inhibitors without the need to grow infectious SARS-CoV.
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(1) Further purification of R17 RNA-dependent RNA polymerase (RNA replicase) of the first DEAE-cellulose fraction resulted in a complete loss of replicase activity, due to the dissociation of replicase into two protein components. This dissociation was effected by treating the enzyme fraction with a high salt concentration, followed by a second DEAE chromatography. One component is eluted from the column at 0.1 M KC1 (ø-factor) and the other at 0.24 M (Fraction DR) which contains functional DNA-dependent RNA polymerase (RNA polymerase, EC 2.7.7.6). The ø-factor does not possess enzymatic activity by itself. (2) Reconstitution of Fraction DR and ø-factor resulted in the restoration of RNA replicase activity and in the loss of RNA polymerase activity. (3) ø-factor was not present in non-infected E. coli, K12, Hfr strain; E. Coli, K12, F-strain; and E. coli B. (4) Fraction DR, on the other hand, could be isolated from these three strains of E. coli without infection. This fraction, which could be derived even from the female strain and contained RNA polymerase activity, catalyzed R17 RNA-directed RNA polymerization in the presence of the ø-factor. From these facts, it was concluded that the ø-factor is coded for by the phage genome and Fraction DR is bacterial proteins, and that the phage specific protein (ø-factor) has a close relationship to the host DNA-dependent RNA polymerase or its subunit(s).
RNA polymerase I
Small nuclear RNA
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Small nuclear RNA
RNA polymerase I
RNA Silencing
Primer (cosmetics)
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The replication of the genomic RNA of the hepatitis C virus (HCV) of positive polarity involves the synthesis of a replication intermediate of negative polarity by the viral RNA-dependent RNA polymerase (NS5B) . In vitro and likely in vivo , the NS5B initiates RNA synthesis without primers. This de novo mechanism needs specific interactions between the polymerase and viral RNA elements . Cis -acting elements involved in the initiation of (–) RNA synthesis have been identified in the 3′ non-coding region and in the NS5B coding region of the HCV RNA. However, the detailed contribution of sequences and/or structures of (–) RNA involved in the initiation of (+) RNA synthesis has been less studied. In this report, we identified an RNA element localized between nucleotides 177 and 222 from the 3′-end of the (–) RNA that is necessary for efficient initiation of RNA synthesis by the recombinant NS5B. By site-directed mutagenesis experiments, we demonstrate that the structure rather than the primary sequence of this domain is important for RNA synthesis. We also demonstrate that the intact structure of this RNA element is also needed for efficient RNA synthesis when the viral NS5B functions in association with other viral and cellular proteins in cultured hepatic cells.
NS5B
Small nuclear RNA
RNA polymerase I
RNA Silencing
Nuclease protection assay
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Replicon
NS5B
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Small nuclear RNA
RNA polymerase I
Nucleoplasm
Precursor mRNA
Five-prime cap
RNA polymerase II
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RNA replication provides a powerful means for the amplification of RNA, but to date it has been found to occur naturally only among RNA viruses. In an attempt to harness this process for the amplification of heterologous mRNAs, both an RNA replicase and its corresponding RNA templates have been expressed in functional form, using vaccinia virus-bacteriophage T7 RNA polymerase vectors. Plasmids were constructed which contained in 5'-to-3' order (i) a bacteriophage T7 promoter; (ii) a full-length cDNA encoding either the RNA replicase (RNA 1) or the coat protein (RNA 2) of flock house virus (FHV), (iii) a cDNA sequence that encoded the self-cleaving ribozyme of satellite tobacco ringspot virus, and (iv) a T7 transcriptional terminator. Both in vitro and in vivo, circular plasmids of this structure were transcribed by T7 RNA polymerase to produce RNAs with sizes that closely resembled those of the two authentic FHV genomic RNAs, RNA 1 and RNA 2. In baby hamster kidney cells that expressed authentic FHV RNA replicase, the RNA 2 (coat protein) transcripts were accurately replicated. Moreover, the RNA 1 (replicase) transcripts directed the synthesis of an enzyme that could replicate not only authentic virion-derived FHV RNA but also the plasmid-derived transcripts themselves. Under the latter conditions, replicative amplification of the RNA transcripts ensued and resulted in a high rate of synthesis of the encoded proteins. This successful expression from a DNA vector of the complex biological process of RNA replication will greatly facilitate studies of its mechanism and is a major step towards the goal of harnessing RNA replication for mRNA amplification.
T7 RNA polymerase
RNA polymerase I
Small nuclear RNA
Replicon
RNA-induced transcriptional silencing
Ligase ribozyme
RNA Silencing
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RNA editing is currently attracting attention as a method for editing genetic information without injury to the genome. The most common approach to edit RNA sequences involves the induction of an A-to-I change by adenosine deaminase acting on RNA (ADAR). However, this method only allows point editing. Here, we report a highly flexible RNA editing method called "RNA overwriting" that employs the influenza A virus RNA-dependent RNA polymerase (RdRp) comprising PA, PB1, and PB2 subunits. RdRp binds to the 5'-cap structure of the host mRNA and cleaves at the AG site, followed by transcription of the viral RNA; this process is called cap-snatching. We engineered a targeting snatch system wherein the target RNA is cleaved and extended at any site addressed by guide RNA (gRNA). We constructed five recombinant RdRps containing a PB2 mutant and demonstrated the editing capability of RdRp mutants by using short RNAs in vitro. PB2-480-containing RdRp exhibited good performance in both cleavage and extension assays; we succeeded in RNA overwriting using PB2-480-containing RdRp. In principle, this method allows RNA editing of any type including mutation, addition, and deletion, by changing the sequence of the template RNA to the sequence of interest; hence, the use of viral RdRp could open new avenues in RNA editing and be a powerful tool in life science.
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Guide RNA
RNA Silencing
Transcription
Small nuclear RNA
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20S RNA is a noninfectious viral single-stranded RNA found in most laboratory strains of the yeast Saccharomyces cerevisiae. 20S RNA encodes a protein of 91 kDa (p91) that contains the common motifs found among RNA-dependent RNA polymerases from RNA viruses. p91 and 20S RNA are noncovalently associated in vivo, forming a ribonucleoprotein complex. We detected an RNA polymerase activity in p91/20S RNA complexes isolated by high-speed centrifugation. The activity was not inhibited by actinomycin D nor alpha-amanitin. The majority of the in vitro products was 20S RNA and the rest was the complementary strands of 20S RNA. Because the extracts were prepared from cells accumulating 20S RNA over its complementary strands, these in vitro products reflect the corresponding activities in vivo. When the p91/20S RNA complexes were subjected to sucrose gradient centrifugation, the polymerase activity cosedimented with the complexes. Furthermore, an RNA polymerase activity was detected in the complex by an antibody-linked polymerase assay using anti-p91 antiserum, suggesting that p91 is present in the active RNA polymerase machinery. These results together indicate that p91 is the RNA-dependent RNA polymerase or a subunit thereof responsible for 20S RNA replication.
RNA polymerase I
Small nuclear RNA
Five-prime cap
RNA polymerase II
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Most Saccharomyces cerevisiae strains carry in their cytoplasm 20 S RNA, a linear single-stranded RNA molecule of 2.5 kilobases in size. 20 S RNA copy number is greatly induced in stress conditions such as starvation, with up to 100,000 copies per cell. 20 S RNA has coding capacity for a protein of 91 kDa (p91) with sequences diagnostic of RNA-dependent RNA polymerases of (+) strand and double-stranded RNA viruses. We detected p91 in 20 S RNA-carrying strains with specific antisera. The amount of p91 in growing cells is higher than that of stationary cells and similar to the one in 20 S RNA-induced cells. Although 20 S RNA is not encapsidated into viral particles, p91 non-covalently forms a ribonucleoprotein complex with 20 S RNA. This suggests a role of p91 in the RNA to RNA synthesis processes required for 20 S RNA replication. Although the strain analyzed also harbors 23 S RNA, a closely related single-stranded RNA, 23 S RNA is not associated with p91 but with its putative RNA polymerase, p104. Similarly, 20 S RNA is not associated with p104 but with p91. These results suggest that 20 S RNA and 23 S RNA replicate independently using their respective cognate RNA polymerases. Most Saccharomyces cerevisiae strains carry in their cytoplasm 20 S RNA, a linear single-stranded RNA molecule of 2.5 kilobases in size. 20 S RNA copy number is greatly induced in stress conditions such as starvation, with up to 100,000 copies per cell. 20 S RNA has coding capacity for a protein of 91 kDa (p91) with sequences diagnostic of RNA-dependent RNA polymerases of (+) strand and double-stranded RNA viruses. We detected p91 in 20 S RNA-carrying strains with specific antisera. The amount of p91 in growing cells is higher than that of stationary cells and similar to the one in 20 S RNA-induced cells. Although 20 S RNA is not encapsidated into viral particles, p91 non-covalently forms a ribonucleoprotein complex with 20 S RNA. This suggests a role of p91 in the RNA to RNA synthesis processes required for 20 S RNA replication. Although the strain analyzed also harbors 23 S RNA, a closely related single-stranded RNA, 23 S RNA is not associated with p91 but with its putative RNA polymerase, p104. Similarly, 20 S RNA is not associated with p104 but with p91. These results suggest that 20 S RNA and 23 S RNA replicate independently using their respective cognate RNA polymerases.
Small nuclear RNA
RNA polymerase I
RNA Silencing
Post-transcriptional modification
Nuclease protection assay
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