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A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans.

Audhya A, Hyndman F, McLeod IX, Maddox AS, Yates JR, Desai A, Oegema K - J. Cell Biol. (2005)

Bottom Line: Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis.This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes.Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA. aaudhya@ucsd.edu

ABSTRACT
Cytokinesis completes cell division and partitions the contents of one cell to the two daughter cells. Here we characterize CAR-1, a predicted RNA binding protein that is implicated in cytokinesis. CAR-1 localizes to germline-specific RNA-containing particles and copurifies with the essential RNA helicase, CGH-1, in an RNA-dependent fashion. The atypical Sm domain of CAR-1, which directly binds RNA, is dispensable for CAR-1 localization, but is critical for its function. Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis. This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes. Depletion of CGH-1 results in sterility, but partially depleted worms produce embryos that exhibit the CAR-1-depletion phenotype. Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.

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CAR-1 copurifies with two widely conserved RNA-binding proteins. (A) Silver stained gel of proteins eluted from S-protein agarose after tandem affinity purification of GFPLAP:CAR-1. Three distinct bands (arrowheads) are present. The two bands labeled with asterisks are contaminants (keratins). (B) Table showing the three proteins identified by solution mass spectrometry. The percent sequence coverage, RNAi phenotype, and molecular weight of each protein is shown. (C) Western blots of CAR-1 immunoprecipitates in the presence (+) or absence (−) of RNaseA, probed with the indicated antibodies. (D) Single sections from deconvolved three-dimensional widefield images of fixed gonads stained for CAR-1 (left column) and PGL-1 (middle column). Merged images with DNA staining (blue) are shown in the right column. Bar, 20 μm. (E) Single section spinning disc confocal imaging of gonads (middle section) from living wild-type or CGH-1–depleted hermaphrodites expressing GFPLAP:CAR-1. Bar, 10 μm.
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fig4: CAR-1 copurifies with two widely conserved RNA-binding proteins. (A) Silver stained gel of proteins eluted from S-protein agarose after tandem affinity purification of GFPLAP:CAR-1. Three distinct bands (arrowheads) are present. The two bands labeled with asterisks are contaminants (keratins). (B) Table showing the three proteins identified by solution mass spectrometry. The percent sequence coverage, RNAi phenotype, and molecular weight of each protein is shown. (C) Western blots of CAR-1 immunoprecipitates in the presence (+) or absence (−) of RNaseA, probed with the indicated antibodies. (D) Single sections from deconvolved three-dimensional widefield images of fixed gonads stained for CAR-1 (left column) and PGL-1 (middle column). Merged images with DNA staining (blue) are shown in the right column. Bar, 20 μm. (E) Single section spinning disc confocal imaging of gonads (middle section) from living wild-type or CGH-1–depleted hermaphrodites expressing GFPLAP:CAR-1. Bar, 10 μm.

Mentions: The above results suggested that CAR-1 interacts with other RNA-binding proteins to execute its function in cytokinesis. To identify such proteins, we used a tandem affinity purification scheme (Cheeseman et al., 2004). Protein complexes containing GFPLAP:CAR-1 were purified by immunoprecipitation with antibodies to GFP, released by cleavage with the tobacco etch virus protease, and reisolated by binding to S-protein agarose. Proteins eluted from the S-protein agarose with urea were analyzed by solution mass spectrometry. Under these stringent purification conditions, we obtained significant sequence coverage for CAR-1 and two additional proteins, the RNA helicase, CGH-1, and the Y-box domain–containing protein, CEY-2, which also is predicted to bind RNA (Fig. 4, A and B).


A complex containing the Sm protein CAR-1 and the RNA helicase CGH-1 is required for embryonic cytokinesis in Caenorhabditis elegans.

Audhya A, Hyndman F, McLeod IX, Maddox AS, Yates JR, Desai A, Oegema K - J. Cell Biol. (2005)

CAR-1 copurifies with two widely conserved RNA-binding proteins. (A) Silver stained gel of proteins eluted from S-protein agarose after tandem affinity purification of GFPLAP:CAR-1. Three distinct bands (arrowheads) are present. The two bands labeled with asterisks are contaminants (keratins). (B) Table showing the three proteins identified by solution mass spectrometry. The percent sequence coverage, RNAi phenotype, and molecular weight of each protein is shown. (C) Western blots of CAR-1 immunoprecipitates in the presence (+) or absence (−) of RNaseA, probed with the indicated antibodies. (D) Single sections from deconvolved three-dimensional widefield images of fixed gonads stained for CAR-1 (left column) and PGL-1 (middle column). Merged images with DNA staining (blue) are shown in the right column. Bar, 20 μm. (E) Single section spinning disc confocal imaging of gonads (middle section) from living wild-type or CGH-1–depleted hermaphrodites expressing GFPLAP:CAR-1. Bar, 10 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171198&req=5

fig4: CAR-1 copurifies with two widely conserved RNA-binding proteins. (A) Silver stained gel of proteins eluted from S-protein agarose after tandem affinity purification of GFPLAP:CAR-1. Three distinct bands (arrowheads) are present. The two bands labeled with asterisks are contaminants (keratins). (B) Table showing the three proteins identified by solution mass spectrometry. The percent sequence coverage, RNAi phenotype, and molecular weight of each protein is shown. (C) Western blots of CAR-1 immunoprecipitates in the presence (+) or absence (−) of RNaseA, probed with the indicated antibodies. (D) Single sections from deconvolved three-dimensional widefield images of fixed gonads stained for CAR-1 (left column) and PGL-1 (middle column). Merged images with DNA staining (blue) are shown in the right column. Bar, 20 μm. (E) Single section spinning disc confocal imaging of gonads (middle section) from living wild-type or CGH-1–depleted hermaphrodites expressing GFPLAP:CAR-1. Bar, 10 μm.
Mentions: The above results suggested that CAR-1 interacts with other RNA-binding proteins to execute its function in cytokinesis. To identify such proteins, we used a tandem affinity purification scheme (Cheeseman et al., 2004). Protein complexes containing GFPLAP:CAR-1 were purified by immunoprecipitation with antibodies to GFP, released by cleavage with the tobacco etch virus protease, and reisolated by binding to S-protein agarose. Proteins eluted from the S-protein agarose with urea were analyzed by solution mass spectrometry. Under these stringent purification conditions, we obtained significant sequence coverage for CAR-1 and two additional proteins, the RNA helicase, CGH-1, and the Y-box domain–containing protein, CEY-2, which also is predicted to bind RNA (Fig. 4, A and B).

Bottom Line: Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis.This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes.Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.

View Article: PubMed Central - PubMed

Affiliation: Ludwig Institute for Cancer Research, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093, USA. aaudhya@ucsd.edu

ABSTRACT
Cytokinesis completes cell division and partitions the contents of one cell to the two daughter cells. Here we characterize CAR-1, a predicted RNA binding protein that is implicated in cytokinesis. CAR-1 localizes to germline-specific RNA-containing particles and copurifies with the essential RNA helicase, CGH-1, in an RNA-dependent fashion. The atypical Sm domain of CAR-1, which directly binds RNA, is dispensable for CAR-1 localization, but is critical for its function. Inhibition of CAR-1 by RNA-mediated depletion or mutation results in a specific defect in embryonic cytokinesis. This cytokinesis failure likely results from an anaphase spindle defect in which interzonal microtubule bundles that recruit Aurora B kinase and the kinesin, ZEN-4, fail to form between the separating chromosomes. Depletion of CGH-1 results in sterility, but partially depleted worms produce embryos that exhibit the CAR-1-depletion phenotype. Cumulatively, our results suggest that CAR-1 functions with CGH-1 to regulate a specific set of maternally loaded RNAs that is required for anaphase spindle structure and cytokinesis.

Show MeSH
Related in: MedlinePlus