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. 2012;7(12):e52447.
doi: 10.1371/journal.pone.0052447. Epub 2012 Dec 20.

The Caenorhabditis Elegans THO Complex Is Required for the Mitotic Cell Cycle and Development

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The Caenorhabditis Elegans THO Complex Is Required for the Mitotic Cell Cycle and Development

Maikel Castellano-Pozo et al. PLoS One. .
Free PMC article

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Abstract

THO is a conserved eukaryotic complex involved in mRNP biogenesis and RNA export that plays an important role in preventing transcription- and RNA-mediated genome instability in mitosis and meiosis. In mammals THO is essential for embryogenesis, which limits our capacity to analyze the physiological relevance of THO during development and in adult organisms. Using Caenorhabditis elegans as a model system we show that the THO complex is essential for mitotic genome integrity and the developmentally regulated mitotic cell cycles occurring during late postembryonic stages.

Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Development defects in thoc-2 mutants.
(A) Representative images of animals of the indicated genotype from the four larva stages and the adult stage at 24 hours post-L4. Scale bars represent 0.1 mm. (B) Body detail of animals of the indicated genotype. The arrow indicates the vulva.
Figure 2
Figure 2. Defective mitosis in thoc-2 mutants.
(A) Representative images of fixed mitotic nuclei from N2(wt) and thoc-2(tm1310) animals immunostained with the mitotic marker CEP-1, and quantification of the mitotic region length. Error bars indicate standard error of mean (n = 15). (B) Poly-A mRNA visualized by FISH using a poly-dT oligonucleotide conjugated with Cy3 in germlines from animals of the indicated genotype. (C) Representative images of fixed mitotic nuclei from animals of the indicated genotype immunostained with GLD-1.
Figure 3
Figure 3. Mitotic DNA damage activation and replication impairment in thoc-2 mutants.
(A) Representative images of a single focal plane through the mitotic region of the germline from N2(wt) and thoc-2(tm1310) counterstained with DAPI, and quantification of the number of mitotic nuclei. Error bars indicate standard errors of means (n = 20). (B) Representative images of fixed mitotic nuclei from animals of the indicated genotype immunostained with antibodies against the replication protein RPA and the checkpoint kinase ATL-1. (C) Representative images of fixed mitotic nuclei from animals of the indicated genotype immunostained with FK-2.
Figure 4
Figure 4. Mitotic replication is impaired in C. elegans thoc-2 germlines and partially alleviated by checkpoint inhibition.
(A) Representative images of fixed germlines of the indicated genotype of adult hermaphrodites 2.5 hours after microinjection with Cy3-dUTP and quantification of Cy3-dUTP incorporation. Error bars indicate standard error of mean (n = 28). Statistically significant differences versus the N2(wt) (p<0.001) is indicated by an asterisk * (Student’s t-Test). (B) Representative images of fixed germlines of the indicated genotype of adult hermaphrodites after microinjection with Cy3-dUTP with or without caffeine and quantification of Cy3-dUTP incorporation. Statistically significant differences versus untreated (p<0.001, n = 15) is indicated by an asterisk * (Student’s t-Test).

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References

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Grant support

This work was supported by grants from the Spanish Ministry of Science and Innovation (BFU2010-16372 and Consolider Ingenio 2010 CSD2007-0015), the European Union (FEDER) and the Junta de Andalucía (CVI4567). MCP was the recipient of a predoctoral training fellowship from the Spanish National Institute of Health Carlos III. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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