Specific targeting of plasmids with Argonaute enables genome editing
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ABSTRACT:
ORGANISM(S): Escherichia coli
PROVIDER: GSE200694 | GEO | 2022/04/17
REPOSITORIES: GEO
Similar Datasets
Project description:Specific targeting of plasmids with Argonaute enables genome editing
| PRJNA826095 | ENA
Project description:Argonaute proteins lie at the heart of the RNA-Induced Silencing Complex (RISC), wherein they use small RNA guides to recognize targets. Some Argonaute proteins can directly cleave their targets, whereas others recruit co-factors to repress independently of “slicing.” Prior studies established the architecture of Argonaute proteins; however, we have not yet had a detailed picture of an Argonaute whose biochemical and biological functions were well established. Here we describe the crystal structure of human Argonaute-2 bound to miR-20a at 2.2 Å resolution. Overall architecture and domain organization is shared with its prokaryotic counterparts, though Ago2 is somewhat more open, with its PAZ domain further removed from the other domains. The miRNA is anchored at both ends by the Mid and PAZ domains but makes several kinks and turns along the binding groove. The RNA confers remarkable stability on the Argonaute protein, locking this otherwise flexible enzyme into a stable conformation. total small RNAs (19-29nt) and small RNAs associated with purified hArgonaute2 purified from SF9 cells. These were processed and sequenced on Illumina GA-II platform.
2012-05-12 | E-GEOD-37931 | biostudies-arrayexpress
Project description:Eukaryotic Argonautes bind small RNAs and use them as guides to find complementary RNA targets and induce gene silencing. Though homologs of eukaryotic Argonautes are present in many bacteria and archaea their small RNA partners and functions are unknown. We found that the Argonaute of Rhodobacter sphaeroides (RsAgo) associates with 15-19 nt small RNAs that correspond to the majority of transcripts. RsAgo also binds single-stranded 22-24 nt DNA molecules that are complementary to the small RNAs and enriched in sequences derived from exogenous plasmids as well as genome-encoded foreign nucleic acids such as transposons and phage genes. Expression of RsAgo in the heterologous E. coli system leads to formation of plasmid–derived small RNA and DNA and plasmid degradation. In a R. sphaeroides mutant lacking RsAgo, expression of plasmid-encoded genes is elevated, while other genes are unaffected. Our results indicate that RNAi-related processes found in eukaryotes are also conserved in bacteria and target foreign nucleic acids.
2013-09-12 | GSE49865 | GEO
Project description:Argonaute proteins lie at the heart of the RNA-Induced Silencing Complex (RISC), wherein they use small RNA guides to recognize targets. Some Argonaute proteins can directly cleave their targets, whereas others recruit co-factors to repress independently of “slicing.” Prior studies established the architecture of Argonaute proteins; however, we have not yet had a detailed picture of an Argonaute whose biochemical and biological functions were well established. Here we describe the crystal structure of human Argonaute-2 bound to miR-20a at 2.2 Å resolution. Overall architecture and domain organization is shared with its prokaryotic counterparts, though Ago2 is somewhat more open, with its PAZ domain further removed from the other domains. The miRNA is anchored at both ends by the Mid and PAZ domains but makes several kinks and turns along the binding groove. The RNA confers remarkable stability on the Argonaute protein, locking this otherwise flexible enzyme into a stable conformation.
2012-05-12 | GSE37931 | GEO
Project description:Eukaryotic Argonautes bind small RNAs and use them as guides to find complementary RNA targets and induce gene silencing. Though homologs of eukaryotic Argonautes are present in many bacteria and archaea their small RNA partners and functions are unknown. We found that the Argonaute of Rhodobacter sphaeroides (RsAgo) associates with 15-19 nt small RNAs that correspond to the majority of transcripts. RsAgo also binds single-stranded 22-24 nt DNA molecules that are complementary to the small RNAs and enriched in sequences derived from exogenous plasmids as well as genome-encoded foreign nucleic acids such as transposons and phage genes. Expression of RsAgo in the heterologous E. coli system leads to formation of plasmid–derived small RNA and DNA and plasmid degradation. In a R. sphaeroides mutant lacking RsAgo, expression of plasmid-encoded genes is elevated, while other genes are unaffected. Our results indicate that RNAi-related processes found in eukaryotes are also conserved in bacteria and target foreign nucleic acids. Small RNA and small DNA from purified RsAgo expressed in Rhodobacter sphaeroides ATCC17025 ("strain 25") and E. coli BL21(DE3) Total small RNA (13-30nt) from R. sphaeroides ATCC17025 and ATCC17029 (wild-type; RsAgo mutant; containing either empty or RsAgo-expressing plasmid) Total ribosomal RNA-depleted RNA from wild type or RsAgo mutant R. sphaeroides ATCC17025
2013-09-12 | E-GEOD-49865 | biostudies-arrayexpress
Project description:Members of the conserved Argonaute protein family use small RNA guides to find their mRNA targets to regulate gene expression and suppress mobile genetic elements in eukaryotes. Argonautes are also present in many bacterial and archaeal species. Unlike eukaryotic proteins, several studied prokaryotic Argonautes use small DNA guides to cleave DNA, a process dubbed DNA interference. However, the natural functions and targets of DNA interference are poorly understood and the mechanisms of DNA guide generation and target discrimination remain unknown. Here, we studied the in vivo activities of a bacterial Argonaute nuclease CbAgo and demonstrated that it induces cleavage of multicopy genetic elements, including plasmids, transposons and repetitive chromosomal loci. Generation of small DNA guides employed by CbAgo requires cooperation between its intrinsic endonuclease activity and the cellular double-strand break repair machinery. The mechanism of guide generation ensures that small DNA guides are enriched in sequences that target foreign DNA and endows CbAgo with capacity to eliminate plasmids and fight phage infection. Similar principles may underlie the specificity of self-nonself discrimination by diverse defense systems in prokaryotes.
2020-07-30 | GSE148594 | GEO
Project description:Members of the conserved Argonaute protein family use small RNA guides to find their mRNA targets to regulate gene expression and suppress mobile genetic elements in eukaryotes. Argonautes are also present in many bacterial and archaeal species. Unlike eukaryotic proteins, several studied prokaryotic Argonautes use small DNA guides to cleave DNA, a process dubbed DNA interference. However, the natural functions and targets of DNA interference are poorly understood and the mechanisms of DNA guide generation and target discrimination remain unknown. Here, we studied the in vivo activities of a bacterial Argonaute nuclease CbAgo and demonstrated that it induces cleavage of multicopy genetic elements, including plasmids, transposons and repetitive chromosomal loci. Generation of small DNA guides employed by CbAgo requires cooperation between its intrinsic endonuclease activity and the cellular double-strand break repair machinery. The mechanism of guide generation ensures that small DNA guides are enriched in sequences that target foreign DNA and endows CbAgo with capacity to eliminate plasmids and fight phage infection. Similar principles may underlie the specificity of self-nonself discrimination by diverse defense systems in prokaryotes.
2020-07-30 | GSE148595 | GEO
Project description:Sequencing iLOV libraries generated from plasmid recombineering
| PRJNA389625 | ENA
Project description:In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15-18 nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of both gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.
2020-03-17 | MSV000085109 | MassIVE
Project description:Critical Assessment of Metagenome Interpretation plasmid raw sequencing data
| PRJEB50299 | ENA