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Eukaryote-Specific Insertion Elements Control Human ARGONAUTE Slicer Activity

ABSTRACT: We solved the crystal structure of human ARGONAUTE1 (hAGO1) bound to endogenous 5'-phosphorylated guide RNAs. To identify structural changes that evolutionarily rendered hAGO1 inactive, we compared our structure with published structures of guide-RNA-containing and cleavage-active hAGO2. Aside from mutation of a catalytic tetrad residue, proline residues at positions 670 and 675 in hAGO1 introduce a kink in the cS7 loop and thereby forming a convex surface within the hAGO1 nucleic-acid-binding channel near the inactive catalytic site. We predicted that even upon restoration of the catalytic tetrad, hAGO1-cS7 sterically hinders the placement of a fully-paired guide-target RNA duplex into the endonuclease active site. Consistent with this hypothesis, reconstitution of the catalytic tetrad (R805H) led to low level hAGO1 cleavage activity, whereas combining R805H with cS7 substitutions (P670S and P675Q) substantially augmented hAGO1 activity. Evolutionary changes to hAGO1 were subtle and readily reversible, suggesting that loading of guide RNA and seed-based miRNA-target RNA pairing constrain its sequence drift. Structure-guided design of mutations that confer slicer activity to hAGO1. RNAs were isolated from recombinant purified hAGO1 and hAGO2, processed from baculovirus-infected Trichoplusia ni insect cells, and then processed for Illumina HiSeq. In addition, total RNA samples from AGO1 baculouvirus infected vs. non-infected Trichoplusia ni cells were isolated and also processed for Illumina HiSeq.

ORGANISM(S): Trichoplusia ni

SUBMITTER: Pavel Morozov

PROVIDER: E-GEOD-48076 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Eukaryote-Specific Insertion Elements Control Human ARGONAUTE Slicer Activity

Project description:We solved the crystal structure of human ARGONAUTE1 (hAGO1) bound to endogenous 5'-phosphorylated guide RNAs. To identify structural changes that evolutionarily rendered hAGO1 inactive, we compared our structure with published structures of guide-RNA-containing and cleavage-active hAGO2. Aside from mutation of a catalytic tetrad residue, proline residues at positions 670 and 675 in hAGO1 introduce a kink in the cS7 loop and thereby forming a convex surface within the hAGO1 nucleic-acid-binding channel near the inactive catalytic site. We predicted that even upon restoration of the catalytic tetrad, hAGO1-cS7 sterically hinders the placement of a fully-paired guide-target RNA duplex into the endonuclease active site. Consistent with this hypothesis, reconstitution of the catalytic tetrad (R805H) led to low level hAGO1 cleavage activity, whereas combining R805H with cS7 substitutions (P670S and P675Q) substantially augmented hAGO1 activity. Evolutionary changes to hAGO1 were subtle and readily reversible, suggesting that loading of guide RNA and seed-based miRNA-target RNA pairing constrain its sequence drift.

2013-06-20 | GSE48076 | GEO

Bacteria-derived small RNAs that co-purify with KpAGO

Project description:The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 Å crystal structure of Kluyveromyces Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1-8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2'-hydroxyl groups pre-organizing the backbone of nucleotides 2-8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide-target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation results and analogies to Ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for cleavage. Employ high-throughput sequencing of small RNAs extracted from soluble or crystalline K. polysporus Ago1(207-1251) that had been purified from E. coli

2012-05-14 | E-GEOD-37724 | biostudies-arrayexpress

Degradome sequencing from S. castellii

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Bacteria-derived small RNAs that co-purify with KpAGO

2012-05-14 | GSE37724 | GEO

Degradome sequencing from S. castellii

Project description:The RNA-induced silencing complex, comprising Argonaute and guide RNA, mediates RNA interference. Here we report the 3.2 M-CM-^E crystal structure of Kluyveromyces Argonaute (KpAGO) fortuitously complexed with guide RNA originating from small-RNA duplexes autonomously loaded by recombinant KpAGO. Despite their diverse sequences, guide-RNA nucleotides 1-8 are positioned similarly, with sequence-independent contacts to bases, phosphates and 2'-hydroxyl groups pre-organizing the backbone of nucleotides 2-8 in a near-A-form conformation. Compared with prokaryotic Argonautes, KpAGO has numerous surface-exposed insertion segments, with a cluster of conserved insertions repositioning the N domain to enable full propagation of guide-target pairing. Compared with Argonautes in inactive conformations, KpAGO has a hydrogen-bond network that stabilizes an expanded and repositioned loop, which inserts an invariant glutamate into the catalytic pocket. Mutation results and analogies to Ribonuclease H indicate that insertion of this glutamate finger completes a universally conserved catalytic tetrad, thereby activating Argonaute for cleavage. High-throughput sequencing of cellular RNAs containing both a 5'-monophosphate group and a poly(A) tail. To identify Argonaute-dependent cleavage tags, sequencing libraries were prepared from M-NM-^Txrn1 and M-NM-^Txrn1 M-NM-^Tago1 strains.

2012-05-14 | E-GEOD-37723 | biostudies-arrayexpress

Protein expression and identification of Tyrp1 Mutant Variants Associated with OCA3 form of albinism.

Project description:Oculocutaneous albinism type 3 (OCA3) is an autosomal recessive disorder caused by mutations in the TYRP1 gene. Tyrosinase-related protein 1 (Tyrp1) is involved in eumelanin synthesis, catalyzing the oxidation of 5,6-dihydroxyindole-2-carboxylic acid oxidase (DHICA) to 5,6-indolequinone-2-carboxylic acid (IQCA). In our work, we are trying to understand the effect of genetic mutations at the level of protein atomic structure and establish a link between the effect of mutations and disease phenotype. Here, for the first time, four OCA3-causing mutations of Tyrp1, C30R, H215Y, D308N, and R326H, were investigated computationally and experimentally to understand Tyrp1 protein stability and catalytic activity. Using the Tyrp1 crystal structure (PDB:5M8L), global mutagenesis was conducted to evaluate mutant protein stability. Consistent with our predictions the foldability parameter, C30R and H215Y should exhibit greater instability, and two other mutants, D308N and R326H, are expected to keep a native conformation. Mass-spectroscopy analysis has confirmed the identity of mutant recombinant variants. SDS-PAGE and Western blot analysis of the purified recombinant proteins confirmed that the foldability parameter correctly predicted the effect of mutations critical for protein stability.

2023-06-12 | PXD034892 | Pride

Protein expression and identification of Tyrp1 Mutant Variants Associated with OCA3 form of albinism.

2023-06-12 | PXD034893 | Pride

Ni Deprivation in Synechococcus WH8102

Project description:Two sets of microarray experiments examining the transcriptional response to Ni deprivation. One set was for growth on ammonium and the other for growth on urea Pairwise replicated comparisons (Ni deprivation in urea and ammonia)

2012-08-08 | E-GEOD-39972 | biostudies-arrayexpress

RNA-Seq of BEAS-2B cells treated with low doses of Ni and NiO nanoparticles as well as NiCl2 for 6 weeks

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The Making of a Slicer: Activation of Human Argonaute-1

Project description:The Argonautes are the central protein component in small RNA silencing pathways. Of the four human Argonautes (hAgo1-4) only hAgo2 is an active slicer. We determined the structure of hAgo1 bound to endogenous copurified RNAs to 1.75 Å resolution and hAgo1 loaded with let-7 miRNA to 2.1 Å. Both structures are strikingly similar to the structures of hAgo2. A conserved DEDH catalytic tetrad within the PIWI domain of hAgo2 is required for its slicing activity. hAgo3, with an intact tetrad, could be activated by swapping in the N domain of hAgo2, without additional PIWI domain mutations. An additional mutation on a loop adjacent to the active site of hAgo1 results in slicer activity that is substantially enhanced by swapping in the N domain of hAgo2. Intriguingly, the elements that make Argonaute an active slicer involve a sophisticated interplay between the active site and more distant regions of the enzyme. RNAsprofiling of small RNAs associated with recombinant protein used for chrystallographic studies

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