HDV RNA Replication: Ancient Relic or Primer?
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Small nuclear RNA
RNA polymerase I
RNA Silencing
Primer (cosmetics)
RNA editing is one of several posttranscriptional events that takes place after a new RNA is synthesized, which can lead to dramatic changes from a gene-encoded origin and affect gene regulation globally. The most widespread type of RNA editing is the conversion of adenosine to inosine (A-to-I) in double-stranded (ds) RNA, which is mediated by the adenosine deaminases acting on RNA (ADAR). The RNA itself plays a role in this regulatory process by forming an assortment of secondary structures such as bulges, stem loops, and hairpins. In some cases, ADAR's action on RNA can change the final protein sequence and function of substrates, giving rise to a greater diversity of proteins than by the DNA-encoded genes. Furthermore, ADAR can reduce the double strandedness of RNA duplexes in the cell, and this can have consequences for gene expression through effects on RNA stability, translational efficiency, and RNA interference (RNAi)-mediated gene silencing pathways. It is apparent that the most extensively edited RNA targets are the noncoding regions of mRNA and in the retrotransposable elements found in these areas. Since dsRNA is a requisite for both ADAR RNA editing and the microRNA pathway, it is now clear that these two different systems are converging for the regulation of gene silencing. Disease associated with ADAR misregulation is beginning to be revealed, and now with new RNA targets identified, it is likely that more will emerge due to the global effects produced by ADAR functions.
ADAR
RNA Silencing
Post-transcriptional regulation
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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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RNA Silencing
RNA virus
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RNA Silencing
Inosine
ADAR
MDA5
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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.
ADAR
Guide RNA
RNA Silencing
Transcription
Small nuclear RNA
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Adenosine-to-inosine (A-to-I) editing is a highly prevalent posttranscriptional modification of RNA, mediated by ADAR (adenosine deaminase acting on RNA) enzymes. In addition to RNA editing, additional functions have been proposed for ADAR1. To determine the specific role of RNA editing by ADAR1, we generated mice with an editing-deficient knock-in mutation (Adar1(E861A), where E861A denotes Glu(861)→Ala(861)). Adar1(E861A/E861A) embryos died at ~E13.5 (embryonic day 13.5), with activated interferon and double-stranded RNA (dsRNA)-sensing pathways. Genome-wide analysis of the in vivo substrates of ADAR1 identified clustered hyperediting within long dsRNA stem loops within 3' untranslated regions of endogenous transcripts. Finally, embryonic death and phenotypes of Adar1(E861A/E861A) were rescued by concurrent deletion of the cytosolic sensor of dsRNA, MDA5. A-to-I editing of endogenous dsRNA is the essential function of ADAR1, preventing the activation of the cytosolic dsRNA response by endogenous transcripts.
ADAR
RNA Silencing
MDA5
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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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Adenosine-to-inosine (A-to-I) editing is a highly prevalent posttranscriptional modification of RNA, mediated by ADAR (adenosine deaminase acting on RNA) enzymes. In addition to RNA editing, additional functions have been proposed for ADAR1. To determine the specific role of RNA editing by ADAR1, we generated mice with an editing-deficient knock-in mutation (Adar1(E861A), where E861A denotes Glu(861)→Ala(861)). Adar1(E861A/E861A) embryos died at ~E13.5 (embryonic day 13.5), with activated interferon and double-stranded RNA (dsRNA)-sensing pathways. Genome-wide analysis of the in vivo substrates of ADAR1 identified clustered hyperediting within long dsRNA stem loops within 3' untranslated regions of endogenous transcripts. Finally, embryonic death and phenotypes of Adar1(E861A/E861A) were rescued by concurrent deletion of the cytosolic sensor of dsRNA, MDA5. A-to-I editing of endogenous dsRNA is the essential function of ADAR1, preventing the activation of the cytosolic dsRNA response by endogenous transcripts.
ADAR
RNA Silencing
MDA5
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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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