The Tetrahymena Argonaute-Binding Protein Giw1p Directs a Mature Argonaute-siRNA Complex to the Nucleus

Noto, Tomoko; Kurth, Henriette; Kataoka, Kensuke; Aronica, Lucia; DeSouza, Leroi V.; Siu, Kin Wai Michael; Pearlman, Ronald E.; Gorovsky, Martin A.; Mochizuki, Kazufumi

doi:10.1016/j.cell.2010.02.010

Cited by 89 publications

(119 citation statements)

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“…The interface itself may also be dynamic during the AGO reaction cycle. This would fit with a flexible role for the AGO proteins, where the adoption of guide and/or target specific conformations (30)(31)(32) would channel them into distinct pathways (such as the miRNA or siRNA pathway).…”

Section: Resultsmentioning

confidence: 99%

Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein

Boland

Huntzinger

Schmidt

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

113

118

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Argonaute proteins (AGOs) are essential effectors in RNAmediated gene silencing pathways. They are characterized by a bilobal architecture, in which one lobe contains the N-terminal and PAZ domains and the other contains the MID and PIWI domains. Here, we present the first crystal structure of the MID-PIWI lobe from a eukaryotic AGO, the Neurospora crassa QDE-2 protein.Compared to prokaryotic AGOs, the domain orientation is conserved, indicating a conserved mode of nucleic acid binding. The PIWI domain shows an adaptable surface loop next to a eukaryote-specific α-helical insertion, which are both likely to contact the PAZ domain in a conformation-dependent manner to sense the functional state of the protein. The MID-PIWI interface is hydrophilic and buries residues that were previously thought to participate directly in the allosteric regulation of guide RNA binding. The interface includes the binding pocket for the guide RNA 5′ end, and residues from both domains contribute to binding. Accordingly, micro-RNA (miRNA) binding is particularly sensitive to alteration in the MID-PIWI interface in Drosophila melanogaster AGO1 in vivo. The structure of the QDE-2 MID-PIWI lobe provides molecular and mechanistic insight into eukaryotic AGOs and has significant implications for understanding the role of these proteins in silencing.P roteins of the Argonaute (AGO) family play essential roles in RNA-mediated gene silencing mechanisms in eukaryotes (1, 2). They are loaded with small noncoding RNAs to form the core of RNA-induced silencing complexes, which repress the expression of target genes at the transcriptional or posttranscriptional level (1, 2). The targets to be silenced are selected through base-pairing interactions between the loaded small RNA (also known as the guide RNA) and an mRNA target containing partially or fully complementary sequences (1-3).Thus far, structural information on full-length AGOs has been available only for the homologous proteins from Archaea and Eubacteria, which preferentially use DNA as a guide (4-10). These studies revealed that AGOs consist of four domains: the N-terminal domain; the PAZ domain, which binds the 3′ end of guide RNAs/DNAs; the MID domain, which provides a binding pocket for the 5′ phosphate of guide RNAs/DNAs; and the PIWI domain, which adopts an RNase H fold and has endonucleolytic activity in some, but not all, AGOs (4-11).For the eukaryotic AGO clade of Argonaute proteins, structural information is available only for the isolated PAZ domains of Drosophila melanogaster (Dm) AGO1 and AGO2, human AGO1 (12-16) and the MID domains of human AGO2 and Neurospora crassa (Nc) QDE-2 (17, 18). Structural information is also available for PAZ domains of the PIWI clade of AGOs (19,20). These studies showed that the PAZ and MID domains of eukaryotic AGOs adopt folds similar to the prokaryotic homologs and recognize the 3′-and 5′-terminal nucleotides of the guide strand, respectively, in a similar manner to their prokaryotic counterparts (12-18).Our previous structure of the isolate...

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Section: Resultsmentioning

confidence: 99%

Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein

Boland

Huntzinger

Schmidt

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

113

118

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“…Cells were fixed and processed as previously described (Kataoka and Mochizuki, 2015 at room temperature for 2 h. They were counterstained with 40 ng/ml DAPI and analyzed using an epifluorescence microscope. DNA-FISH was performed as previously described (Loidl and Scherthan, 2004;Noto et al, 2010).…”

Section: Immunofluorescence Staining and Dna-fishmentioning

confidence: 99%

“…The first group consists of proteins required for the formation of heterochromatin at IESs, including the RNAi machinery components Dcl1p, Twi1p, Giw1p and Ema1p, the histone methyltransferase Ezl1p and the HP1-like protein Pdd1p (Aronica et al, 2008;Coyne et al, 1999;Liu et al, 2004Liu et al, , 2007Malone et al, 2005;Mochizuki and Gorovsky, 2005;Noto et al, 2010). The second group includes proteins that are dispensable for heterochromatin formation but are required for its assembly into heterochromatin bodies: Jub1p, Lia1p, Lia4p, Lia5p, Pdd2p, Tku80p and Tpb2p Rexer and Chalker, 2007;Horrell and Chalker, 2014;Shieh and Chalker, 2013;Liu et al, 2007;Cheng et al, 2010;Lin et al, 2012;Vogt and Mochizuki, 2013;Kataoka and Mochizuki, 2015).…”

Section: Introductionmentioning

confidence: 99%

Heterochromatin aggregation during DNA elimination in Tetrahymena is facilitated by a prion-like protein

Kataoka

Mochizuki

2017

Journal of Cell Science

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Regulated aggregations of prion and prion-like proteins play physiological roles in various biological processes. However, their structural roles in the nucleus are poorly understood. Here, we show that the prion-like protein Jub6p is involved in the regulation of chromatin structure in the ciliated protozoan Tetrahymena thermophila. Jub6p forms sodium dodecyl sulfate (SDS)-resistant aggregates when it is ectopically expressed in vegetative cells and binds to RNA in vitro. Jub6p is a heterochromatin component and is important for the formation of heterochromatin bodies during the process of programmed DNA elimination. We suggest that RNAprotein aggregates formed by Jub6p are an essential architectural component for the assembly of heterochromatin bodies.

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Section: Strains and Culture Conditionsmentioning

confidence: 99%

“…Combinations of KO strains that mate were determined by directly checking their mating ability. DNA elimination of the Tlr1 element was analyzed by DNA FISH, as described previously (Noto et al 2010).Complementation assay pHA-pur4 was produced by replacing the neo gene of pHAneo4 (Kataoka et al 2010) with the puromycin-resistance gene (pac) of the pur4 cassette (Iwamoto et al 2014). The pur4 cassette expresses a puromycin-resistance gene in the presence of cadmium ions.…”

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confidence: 99%

Targeted Gene Disruption by Ectopic Induction of DNA Elimination in Tetrahymena

Hayashi¹,

Mochizuki

2015

Genetics

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Tetrahymena is a useful eukaryotic model for biochemistry and molecular cell biology studies. We previously demonstrated that targeted ectopic DNA elimination, also called co-Deletion (coDel), can be induced by the introduction of an internal eliminated sequence (IES)-target DNA chimeric construct. In this study, we demonstrate that coDel occurs at most of the loci tested and can be used for the production of somatic gene KO strains. We also showed that coDel at two loci can be simultaneously induced by a single transformation; thus, coDel can be used to disrupt multiple gene loci in a single cell. Therefore, coDel is a useful tool for functional genetics in Tetrahymena and further extends the usefulness of this model organism.KEYWORDS Tetrahymena; DNA elimination; RNAi; gene knockout T HE ciliated protozoan Tetrahymena thermophila can grow at an exceptionally high rate (its doubling time is approximately 2 hr) and can reach a high density (a few million cells per milliliter) under simple and inexpensive culture conditions (reviewed in Orias et al. 2000). In combination with robust genetic manipulation methods (reviewed in Chalker 2012), Tetrahymena is a useful model eukaryote for biochemistry and molecular cell biology studies (reviewed in Collins and Gorovsky 2005).Three strategies for loss-of-function genetic studies have been established for this organism. The first strategy is a germline knockout (KO), in which one of the two gene copies in the diploid micronucleus (MIC) is replaced with a drug-resistance gene by homologous recombination, and two heterozygous strains are then sexually crossed to obtain a homozygous germline KO strain (Cassidy-Hanley et al. 1997). The second strategy is somatic KO, in which one of the 45 copies of a gene in the polyploid macronucleus (MAC) is replaced with a drugresistance gene by homologous recombination, and "phenotypic assortment" is used to obtain cells with the drug-resistance gene at all loci (Merriam and Bruns 1988). Phenotypic assortment is possible because MAC chromosomes are randomly segregated into daughter cells in vegetative cell division and a stepwise increase of the drug in culture selects for cells that have more drug-resistance genes. The final strategy is gene knockdown (KD) by RNA interference (RNAi), in which a construct expressing long hairpin RNA that is complementary to the gene of interest is introduced into a nonessential locus in the MAC and small RNAs produced from the hairpin RNA posttranscriptionally silence the expression of the gene (HowardTill and Yao 2006).Although the first two gene KO strategies have been reliably used to study individual gene functions, they are not suitable for high-throughput genetic screening because the integration of a KO construct into the MIC germline occurs at very low efficiency and the phenotypic assortment process used in somatic KOs require the careful control of drug concentrations in each strain and culture step. Moreover, it takes 1 month to obtain KO strains using these methods. For germline KO, t...

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The Tetrahymena Argonaute-Binding Protein Giw1p Directs a Mature Argonaute-siRNA Complex to the Nucleus

Cited by 89 publications

References 46 publications

Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein

Crystal structure of the MID-PIWI lobe of a eukaryotic Argonaute protein

Heterochromatin aggregation during DNA elimination in Tetrahymena is facilitated by a prion-like protein

Targeted Gene Disruption by Ectopic Induction of DNA Elimination in Tetrahymena

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