Protection of specific maternal messenger RNAs by the P body protein CGH-1 (Dhh1/RCK) during Caenorhabditis elegans oogenesis - PubMed (original) (raw)
Protection of specific maternal messenger RNAs by the P body protein CGH-1 (Dhh1/RCK) during Caenorhabditis elegans oogenesis
Peter R Boag et al. J Cell Biol. 2008.
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.
Figures
Figure 1.
A C. elegans hermaphrodite gonad, illustrating models discussed in the text. Germ cells develop in an “assembly line” fashion from stem cells that are regulated by the somatic distal tip cell (Hubbard and Greenstein, 2005). Each of two gonad arms produces sperm during the fourth larval stage, then oocytes during adulthood. Germ cells initially share a common cytoplasm, which flows as shown by arrows (Wolke et al., 2007). Transcription increases sharply upon progression through the transition zone and entry into meiosis I. As new mRNAs move into the syncytial gonad core, some appear to pass through germ (P) granules (inset, green), which are located just outside of most nuclear pores (Pitt et al., 2000; Schisa et al., 2001). CGH-1 associates with and protects particular translationally regulated maternal mRNAs in the context of storage bodies, which may be assembled on germ (P) granules (inset). Oocytes enlarge and become cellularized in the proximal region, and fertilization occurs in the spermatheca. Transcription ceases as oocytes enter diakinesis (Walker et al., 2007). Further development depends upon translationally regulated maternal gene products until embryonic transcription begins at the four-cell stage (Seydoux and Dunn, 1997). CGH-1 then associates with PATR-1 in _patr-1_–dependent somatic P bodies (red dots).
Figure 2.
Somatic embryonic CGH-1 foci have characteristics of decapping-associated P bodies. (A) Embryonic founder cells. Four successive asymmetrical divisions of the germ cell precursor each produce a somatic and germ cell daughter. The founding germ line cell P4 gives rise to the germ cell precursors Z2 and Z3 before the 200-cell stage. (B) Effects of dcap-1,-2 and patr-1 RNAi on embryonic CGH-1 foci. Embryos were immunostained for CGH-1 and then analyzed by confocal microscopy. Z series projections of staining throughout the embryo are shown. In germ cell precursors (posteriorly and to the right, outlined by broken lines), most CGH-1 particles colocalize with germ (P) granules (Navarro et al., 2001). At the two- and four-cell stages, CGH-1 foci are distributed similarly in N2 (WT), dcap-1/-2(RNAi), and patr-1(RNAi) embryos. At later stages, in somatic cells, CGH-1 foci are much more prominent in dcap-1/-2(RNAi) embryos than in N2, and are almost absent in patr-1(RNAi) embryos. In each experimental set, >50 embryos were examined at each stage shown. (C) Detailed images of somatic P bodies in 50-cell embryos, corresponding to the boxes in B. (D) Quantitation of somatic embryonic P bodies after dcap-1,-2 or patr-1 RNAi. CGH-1 foci were quantitated in confocal z series projections, with foci of fewer than six pixels and germ (P) granules excluded. Data points each correspond to two-to-seven embryos. (E) Effects of dcap-1,-2 and patr-1 RNAi on embryonic CGH-1∷GFP foci. Single plane differential interference contrast (DIC) and fluorescent images are shown of representative ∼30-cell embryos from a rescuing CGH-1∷GFP transgenic strain. CGH-1∷GFP foci in germ (P) granules were unaffected by these RNAi treatments, but somatic foci were more prominent in decapping-defective embryos than WT, and rarely visible in patr-1(RNAi) embryos. In each set, >40 embryos were examined at this and other early embryonic stages. Bars: (B) 10 μm; (C) 5 μm; (E), 10 μm.
Figure 3.
Association of CGH-1 and PATR-1 in somatic P bodies. (A) Somatic P bodies in dcap-2(tm2470) larvae. Single-plane confocal images reveal colocalizing CGH-1 (green) and PATR-1(N)-staining (red) particles in many somatic cells in dcap-2(tm2470) but not N2 (WT) L1-stage larvae (outlined by broken lines). Regions outlined by dashed boxes are enlarged on the far right. n > 100 for each set. Bars: 10 μm; (far right) 2 μm. (B) PATR-1(N) staining marks somatic P bodies in the embryo. WT embryos were analyzed by antibody staining and confocal microscopy (z series projections are shown). Germ cell precursors are outlined by broken lines, and in eight-cell embryos, the somatic sister of P3 (C blastomere) is similarly delineated. Robust PATR-1(N) staining is first detected in four-cell embryos, in which it colocalizes with CGH-1 in somatic blastomeres. After each successive division of the germ cell precursor PATR-1(N), staining is initially low, then increases. These low levels are apparent in the EMS blastomere (detail of the boxed area shown on the right), and also in the C blastomere (sister of P3) at the eight-cell stage. More than 50 embryos in each set were examined in each of at least two experiments. Bars: 10 μm; (far right) 2 μm. (C) CGH-1 and PATR-1 levels in N2, dcap-2(tm2470), and patr-1(2402) adult hermaphrodites. PATR-1 was detected with anti–PATR-1(N). (D) PATR-1 expression in N2 and glp-4(bn2) adults, which essentially lack germ cells when grown at the restrictive temperature of 25°C. Densitometry indicated that glp-4(bn2) adult PATR-1 levels were ∼68% of WT. (E) PATR-1 coIPs with CGH-1. CGH-1 IPs from 2.5 mg of adult (A) and embryo (Em) protein extracts were Western blotted for PATR-1.
Figure 4.
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
).
Figure 5.
CGH-1 affects mRNA localization during oogenesis. (A–C) CGH-1 and SL1 leader sequence localization in proximal oocytes in fog-2(q71) females. Extruded gonads from 1-d-old adults were stained for CGH-1 and analyzed by in situ hybridization to an Alexa 488–linked SL1 antisense probe. SL1-containing mRNA accumulates in large foci that colocalize with CGH-1 and in separate small puncta. In parallel, an SL1 sense probe showed little or no diffuse signal (not depicted). Oocytes are outlined by broken lines. n > 60. (D and E) SL1 localization in WT hermaphrodite gonads analyzed by in situ hybridization. (F–I) Abnormal mRNA localization during oogenesis in cgh-1(ok492). Extruded hermaphrodite gonads were analyzed by in situ hybridization for SL1 (green) and staining with anti–CAR-1 (red) and DAPI. The transition zone and pachytene region of a typical gonad are shown, with distal toward the top right. CAR-1 and some SL1-containing mRNAs colocalize in sheetlike structures. The bulbous shape of this gonad is characteristic of many animals that lack CGH-1 (Navarro et al., 2001). Confocal z series projections are shown. For D–I, n > 60 for WT and cgh-1(ok492). Bars, 10 μm.
Figure 6.
CGH-1 binds and stabilizes particular mRNAs during oogenesis. (A) Analysis of mRNA abundance and distribution by in situ hybridization. Probes were hybridized to dissected gonads from 1-d-old N2 and cgh-1(ok492) animals. For each probe and genotype tested, at least 20–25 gonads were examined in each of two or more experiments. Six of seven mRNAs that were not associated with CGH-1 did not differ detectably between N2 and cgh-1 mutant animals (Fig. S3, C and D, available at
http://www.jcb.org/cgi/content/full/jcb.200801183/DC1
). In contrast, all six CGH-1–associated mRNAs analyzed were present at markedly lower levels in cgh-1(ok492) gonads. Distal is shown to the right (asterisks). Each in situ hybridization experiment gave consistent results among gonads examined, for which representative images are shown. Bar, 50 μm. (B) Relative abundance of CGH-1–associated and unassociated mRNAs in cgh-1(ok492) adults compared with WT, assayed by RT-qPCR. Each mRNA value was normalized to actin (act-1), and WT levels were converted to 1. The mean of three biological experiments is graphed. (C) Relative abundance of CGH-1–associated and unassociated mRNAs in fog-2(q71) adults compared with WT. Data are from two independent experiments. Error bars indicate SEM. (D) A model for germ line CGH-1 functions. Many maternal mRNAs rely on the CGH-1 complex to protect them from degradation and possibly for translational silencing. Some CGH-1–associated mRNAs are known to be translationally regulated through binding of sequence-specific proteins to their 3′ UTRs (see text). Such proteins might be present on individual mRNAs in conjunction with the CGH-1 complex or could assume control of these mRNAs after they are released by this complex.
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