A
            <i>Tetrahymena</i>
            Hsp90 co‐chaperone promotes si
            <scp>RNA</scp>
            loading by
            <scp>ATP</scp>
            ‐dependent and
            <scp>ATP</scp>
            ‐independent mechanisms

Woehrer, Sophie L; Aronica, Lucia; Suhren, Jan H.; Busch, Clara Jana-Lui; Noto, Tomoko; Mochizuki, Kazufumi

doi:10.15252/embj.201490062

Cited by 26 publications

(25 citation statements)

References 61 publications

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“…Data represents the average of the results obtained by Gorowski, Miao and Pearlman Labs (Miao et al, 2009). *Note that TTHERM_00526270 contains two genes; one COI3 (transcript id: gene_000015720), and the other, Atg4 (C54 peptidase domain, transcript id: gene_000006383) (Woehrer et al, 2015). We focused on the latter transcript in this study.…”

Section: Resultsmentioning

confidence: 99%

A comparative in-silico analysis of autophagy proteins in ciliates

Aslan

Küçükoğlu

Arslanyolu

2017

PeerJ

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Autophagy serves as a turnover mechanism for the recycling of redundant and/or damaged macromolecules present in eukaryotic cells to re-use them under starvation conditions via a double-membrane structure known as autophagosome. A set of eukaryotic genes called autophagy-related genes (ATGs) orchestrate this highly elaborative process. The existence of these genes and the role they play in different eukaryotes are well-characterized. However, little is known of their role in some eukaryotes such as ciliates. Here, we report the computational analyses of ATG genes in five ciliate genomes to understand their diversity. Our results show that Oxytricha trifallax is the sole ciliate which has a conserved Atg12 conjugation system (Atg5-Atg12-Atg16). Interestingly, Oxytricha Atg16 protein includes WD repeats in addition to its N-terminal Atg16 domain as is the case in multicellular organisms. Additionally, phylogenetic analyses revealed that E2-like conjugating protein Atg10 is only present in Tetrahymena thermophila. We fail to find critical autophagy components Atg5, Atg7 and Atg8 in the parasitic ciliate Ichthyophthirius multifiliis. Contrary to previous reports, we also find that ciliate genomes do not encode typical Atg1 since all the candidate sequences lack an Atg1-specific C-terminal domain which is essential for Atg1 complex formation. Consistent with the absence of Atg1, ciliates also lack other members of the Atg1 complex. However, the presence of Atg6 in all ciliates examined here may rise the possibility that autophagosome formation could be operated through Atg6 in ciliates, since Atg6 has been shown as an alternative autophagy inducer. In conclusion, our results highlight that Atg proteins are partially conserved in ciliates. This may provide a better understanding for the autophagic destruction of the parental macronucleus, a developmental process also known as programmed nuclear death in ciliates.

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Section: Resultsmentioning

confidence: 99%

A comparative in-silico analysis of autophagy proteins in ciliates

Aslan

Küçükoğlu

Arslanyolu

2017

PeerJ

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“…The open reading frames of TTHERM_01415190 (JUB6), TTHERM_ 00647510, TTHERM_00145310, TTHERM_00421130, JUB1, LIA1, LIA4, LIA5, PDD1 and PDD2 were PCR amplified from Tetrahymena cDNA and cloned into pBNMB1-HA (Woehrer et al, 2015). The DNA oligonucleotides used to generate the constructs are listed in Table S1.…”

Section: Ectopic Expression Of Proteins In Vegetative Cellsmentioning

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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“…The clones 6-1, 9-2, 9-6, 17-1 of TTHERM_00301940 KO cells were selected for further study. TTHERM_00301940 is exclusively expressed during conjugation and many conjugation-specific genes are required for DNA elimination (Mochizuki et al 2002;Taverna et al 2002;Liu et al 2007;Cheng et al 2010;Shieh and Chalker 2013;Woehrer et al 2015); we sought to determine whether TTHERM_00301940 is also involved in DNA elimination. TTHERM_00301940 KO strains (6-1 3 9-6 [= cross 1 in Table 1] or 9-2 3 17-1 [= cross 2 in Table 1]) were mated, and the DNA elimination in their exconjugants (progeny) was analyzed by DNA FISH using probes complementary to the Tlr1 element.…”

Section: Two-target Loci Can Be Simultaneously Deleted By Codelmentioning

confidence: 99%

“…For this purpose, we asked whether some of the early conjugation-specific genes subjected to coDel (Figure 1, E-U) are important for programmed DNA elimination, such as DED1. In addition, we studied two early conjugation-specific genes [TTHERM_00079530 (also called COI5; Woehrer et al 2015) and TTHERM_00313180] that we subjected to coDel in our previous study . For TTHERM_00285420, TTHERM_00460720, and TTHERM_00675560, the original coDel attempts with 600-to 700-bp targets (Figure 1, F, I, and N) did not provide sufficient DNA elimination to produce KO strains.…”

Section: Establishment Of a Complementation Assay Systemmentioning

confidence: 99%

“…Because the KO cells for TTHERM_00079530, TTHERM_00112830, TTHERM_01044400, TTHERM_00494510, and TTHERM_00675560 did not produce exconjugants for unknown reasons, we could not check whether these genes played any role in DNA elimination. Two of 11 conjugation-specific genes were also determined as genes required for completing conjugation in our previous conventional homologous recombination-based gene KO studies (Woehrer et al 2015). Because conjugation involves multiple complex processes, it is not surprising that many conjugationspecific genes are required for producing exconjugants.…”

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confidence: 99%

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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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A Tetrahymena Hsp90 co‐chaperone promotes si RNA loading by ATP ‐dependent and ATP ‐independent mechanisms

Cited by 26 publications

References 61 publications

A comparative in-silico analysis of autophagy proteins in ciliates

A comparative in-silico analysis of autophagy proteins in ciliates

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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