Limits...
Protection of specific maternal messenger RNAs by the P body protein CGH-1 (Dhh1/RCK) during Caenorhabditis elegans oogenesis.

Boag PR, Atalay A, Robida S, Reinke V, Blackwell TK - J. Cell Biol. (2008)

Bottom Line: In contrast, during oogenesis, CGH-1 forms patr-1-independent mRNA storage bodies.CGH-1 then associates with translational regulators and a specific set of maternal mRNAs, and prevents those mRNAs from being degraded.Our results identify somatic and germ cell CGH-1 functions that are distinguished by the involvement of PATR-1, and reveal that during oogenesis, numerous translationally regulated mRNAs are specifically protected by a CGH-1-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Joslin Diabetes Center, Harvard Stem Cell Institute, and 2Department of Pathology, Harvard Medical School, Boston, MA 02215, USA.

ABSTRACT
During oogenesis, numerous messenger RNAs (mRNAs) are maintained in a translationally silenced state. In eukaryotic cells, various translation inhibition and mRNA degradation mechanisms congregate in cytoplasmic processing bodies (P bodies). The P body protein Dhh1 inhibits translation and promotes decapping-mediated mRNA decay together with Pat1 in yeast, and has been implicated in mRNA storage in metazoan oocytes. Here, we have investigated in Caenorhabditis elegans whether Dhh1 and Pat1 generally function together, and how they influence mRNA sequestration during oogenesis. We show that in somatic tissues, the Dhh1 orthologue (CGH-1) forms Pat1 (patr-1)-dependent P bodies that are involved in mRNA decapping. In contrast, during oogenesis, CGH-1 forms patr-1-independent mRNA storage bodies. CGH-1 then associates with translational regulators and a specific set of maternal mRNAs, and prevents those mRNAs from being degraded. Our results identify somatic and germ cell CGH-1 functions that are distinguished by the involvement of PATR-1, and reveal that during oogenesis, numerous translationally regulated mRNAs are specifically protected by a CGH-1-dependent mechanism.

Show MeSH

Related in: MedlinePlus

CGH-1 associates with specific maternal mRNAs. (A) Identification of CGH-1–associated mRNAs by RIP-Chip. CGH-1 was immunoprecipitated from extracts from 1-d-old adult hermaphrodites, then mRNA was extracted from material that was eluted by the immunogenic CGH-1 peptide, a protocol that isolates the CGH-1 complex (Boag et al., 2005). The control was rabbit IgG. After linear amplification, samples were labeled with Cy3 or Cy5 and hybridized to microarrays. Fold enrichment was averaged from four RIP-Chip experiments. (B) Expression profiles of CGH-1–enriched mRNAs. The vast majority of annotated CGH-1–enriched mRNAs are expressed primarily in adult hermaphrodite gonads, which produce only oocytes (Reinke et al., 2000, 2004). (C) CoIP with CGH-1 does not correlate with abundance. The relative enrichment in CGH-1 IPs and the number of nonambiguous SAGE tags (normalized to 100,000) are plotted for the 50 mRNAs that were most enriched in CGH-1 IPs (blue) and the 50 most abundant mRNAs (red) from a dissected gonad SAGE library (SW040; http://tock.bcgsc.bc.ca/cgi-bin/sage140).
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
getmorefigures.php?uid=PMC2500139&req=5

fig4: CGH-1 associates with specific maternal mRNAs. (A) Identification of CGH-1–associated mRNAs by RIP-Chip. CGH-1 was immunoprecipitated from extracts from 1-d-old adult hermaphrodites, then mRNA was extracted from material that was eluted by the immunogenic CGH-1 peptide, a protocol that isolates the CGH-1 complex (Boag et al., 2005). The control was rabbit IgG. After linear amplification, samples were labeled with Cy3 or Cy5 and hybridized to microarrays. Fold enrichment was averaged from four RIP-Chip experiments. (B) Expression profiles of CGH-1–enriched mRNAs. The vast majority of annotated CGH-1–enriched mRNAs are expressed primarily in adult hermaphrodite gonads, which produce only oocytes (Reinke et al., 2000, 2004). (C) CoIP with CGH-1 does not correlate with abundance. The relative enrichment in CGH-1 IPs and the number of nonambiguous SAGE tags (normalized to 100,000) are plotted for the 50 mRNAs that were most enriched in CGH-1 IPs (blue) and the 50 most abundant mRNAs (red) from a dissected gonad SAGE library (SW040; http://tock.bcgsc.bc.ca/cgi-bin/sage140).

Mentions: Interactions between CGH-1 and these proteins were abolished by RNase treatment, which suggests that mRNA might be present in this germ line CGH-1 complex (Boag et al., 2005). To test this idea, we performed four experiments in which we used RNA IP and microarray analysis (RIP-Chip) to detect mRNAs that coIP with CGH-1 (Fig. 4 A). We performed these IPs using extracts that were prepared from intact 1-d-old adults in a way that excluded embryos so that they were enriched for CGH-1 that is expressed in adult germ cells. We then identified mRNAs that were on average at least threefold overrepresented in CGH-1 IPs relative to IgG controls because we expected that this arbitrarily defined level would identify specific interactions. Surprisingly, only 392 mRNAs met this criterion (Fig. 4 B, and Tables II and S2, available at http://www.jcb.org/cgi/content/full/jcb.200801183/DC1).


Protection of specific maternal messenger RNAs by the P body protein CGH-1 (Dhh1/RCK) during Caenorhabditis elegans oogenesis.

Boag PR, Atalay A, Robida S, Reinke V, Blackwell TK - J. Cell Biol. (2008)

CGH-1 associates with specific maternal mRNAs. (A) Identification of CGH-1–associated mRNAs by RIP-Chip. CGH-1 was immunoprecipitated from extracts from 1-d-old adult hermaphrodites, then mRNA was extracted from material that was eluted by the immunogenic CGH-1 peptide, a protocol that isolates the CGH-1 complex (Boag et al., 2005). The control was rabbit IgG. After linear amplification, samples were labeled with Cy3 or Cy5 and hybridized to microarrays. Fold enrichment was averaged from four RIP-Chip experiments. (B) Expression profiles of CGH-1–enriched mRNAs. The vast majority of annotated CGH-1–enriched mRNAs are expressed primarily in adult hermaphrodite gonads, which produce only oocytes (Reinke et al., 2000, 2004). (C) CoIP with CGH-1 does not correlate with abundance. The relative enrichment in CGH-1 IPs and the number of nonambiguous SAGE tags (normalized to 100,000) are plotted for the 50 mRNAs that were most enriched in CGH-1 IPs (blue) and the 50 most abundant mRNAs (red) from a dissected gonad SAGE library (SW040; http://tock.bcgsc.bc.ca/cgi-bin/sage140).
© Copyright Policy
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2500139&req=5

fig4: CGH-1 associates with specific maternal mRNAs. (A) Identification of CGH-1–associated mRNAs by RIP-Chip. CGH-1 was immunoprecipitated from extracts from 1-d-old adult hermaphrodites, then mRNA was extracted from material that was eluted by the immunogenic CGH-1 peptide, a protocol that isolates the CGH-1 complex (Boag et al., 2005). The control was rabbit IgG. After linear amplification, samples were labeled with Cy3 or Cy5 and hybridized to microarrays. Fold enrichment was averaged from four RIP-Chip experiments. (B) Expression profiles of CGH-1–enriched mRNAs. The vast majority of annotated CGH-1–enriched mRNAs are expressed primarily in adult hermaphrodite gonads, which produce only oocytes (Reinke et al., 2000, 2004). (C) CoIP with CGH-1 does not correlate with abundance. The relative enrichment in CGH-1 IPs and the number of nonambiguous SAGE tags (normalized to 100,000) are plotted for the 50 mRNAs that were most enriched in CGH-1 IPs (blue) and the 50 most abundant mRNAs (red) from a dissected gonad SAGE library (SW040; http://tock.bcgsc.bc.ca/cgi-bin/sage140).
Mentions: Interactions between CGH-1 and these proteins were abolished by RNase treatment, which suggests that mRNA might be present in this germ line CGH-1 complex (Boag et al., 2005). To test this idea, we performed four experiments in which we used RNA IP and microarray analysis (RIP-Chip) to detect mRNAs that coIP with CGH-1 (Fig. 4 A). We performed these IPs using extracts that were prepared from intact 1-d-old adults in a way that excluded embryos so that they were enriched for CGH-1 that is expressed in adult germ cells. We then identified mRNAs that were on average at least threefold overrepresented in CGH-1 IPs relative to IgG controls because we expected that this arbitrarily defined level would identify specific interactions. Surprisingly, only 392 mRNAs met this criterion (Fig. 4 B, and Tables II and S2, available at http://www.jcb.org/cgi/content/full/jcb.200801183/DC1).

Bottom Line: In contrast, during oogenesis, CGH-1 forms patr-1-independent mRNA storage bodies.CGH-1 then associates with translational regulators and a specific set of maternal mRNAs, and prevents those mRNAs from being degraded.Our results identify somatic and germ cell CGH-1 functions that are distinguished by the involvement of PATR-1, and reveal that during oogenesis, numerous translationally regulated mRNAs are specifically protected by a CGH-1-dependent mechanism.

View Article: PubMed Central - PubMed

Affiliation: Joslin Diabetes Center, Harvard Stem Cell Institute, and 2Department of Pathology, Harvard Medical School, Boston, MA 02215, USA.

ABSTRACT
During oogenesis, numerous messenger RNAs (mRNAs) are maintained in a translationally silenced state. In eukaryotic cells, various translation inhibition and mRNA degradation mechanisms congregate in cytoplasmic processing bodies (P bodies). The P body protein Dhh1 inhibits translation and promotes decapping-mediated mRNA decay together with Pat1 in yeast, and has been implicated in mRNA storage in metazoan oocytes. Here, we have investigated in Caenorhabditis elegans whether Dhh1 and Pat1 generally function together, and how they influence mRNA sequestration during oogenesis. We show that in somatic tissues, the Dhh1 orthologue (CGH-1) forms Pat1 (patr-1)-dependent P bodies that are involved in mRNA decapping. In contrast, during oogenesis, CGH-1 forms patr-1-independent mRNA storage bodies. CGH-1 then associates with translational regulators and a specific set of maternal mRNAs, and prevents those mRNAs from being degraded. Our results identify somatic and germ cell CGH-1 functions that are distinguished by the involvement of PATR-1, and reveal that during oogenesis, numerous translationally regulated mRNAs are specifically protected by a CGH-1-dependent mechanism.

Show MeSH
Related in: MedlinePlus