Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans - PubMed (original) (raw)
Multiple potential germ-line helicases are components of the germ-line-specific P granules of Caenorhabditis elegans
M E Gruidl et al. Proc Natl Acad Sci U S A. 1996.
Abstract
Two components of the germ-line-specific P granules of the nematode Caenorhabditis elgans have been identified using polyclonal antibodies specific for each. Both components are putative germ-line RNA helicases (GLHs) that contain CCHC zinc fingers of the type found in the RNA-binding nucleocapsid proteins of retroviruses. The predicted GLH-1 protein has four CCHC fingers; GLH-2 has six. Both GLH proteins localize in the P granules at all stage of germ-line development. However, the two glh genes display different patterns of RNA and protein accumulation in the germ lines of hermaphrodites and males. Injection of antisense glh-1 or glh-2 RNA into wild-type worms causes some offspring to develop into sterile adults, suggesting that either or both genes are required for normal germ-line development. As these very similar glh genes physically map within several hundred kilobases of one another, it seems likely that they represent a fairly recent gene duplication event.
Figures
Figure 1
Alignment of GLH-1 and GLH-2. N-terminal glycine-rich imperfect repeats are shaded in grey. CCHC zinc fingers are indicated with black backing. The zinc finger consensus in the two GLH proteins is PxxCFNCxxxGHRSxxCPEP; these amino acids are found in at least 7/10 fingers. Comparisons of specific zinc fingers are shown at the bottom with differences in black. The conserved motifs found in all DEAD-box RNA helicases (26) are boxed. The 22-amino acid peptides used to produce GLH-1 and GLH-2-specific peptides are underlined (N termini). Sites of introns in the corresponding glh-1 and glh-2 genomic sequences are indicated with arrowheads. The 58 amino acids encoded by a 168-nt internal_Bam_HI–_Bam_HI fragment were absent in the original report of the glh-1 cDNA (18); the corrected sequence is GenBank accession no. L19948L19948.
Figure 2
In situ hybridization to C. elegans adults and embryos. glh-1 hybridization to a splayed hermaphrodite (A) and male (B) using a 253-nt antisense probe from the glh-1_-specific 5′_Eco_RI–_Bam_HI fragment (18). (C) Sense strand of the same glh-1 probe as a negative control. The gonads of the splayed worms are indicated with arrowheads. The 4′,6-diamidino-2-phenylindole (DAPI)-stained nuclei are blue. All glh-1 slides were hybridized with 5 × 105 dpm and exposed for 7 days. (D and_E) Antisense glh-2 RNA hybridization to a hermaphrodite (D) and male (E). The probe used was 340 bp long, including 130 bp of the 3′-most coding region and the entire 210-bp glh-2 3′ UTR, minus the poly(A) tail. This probe was determined to be specific for glh-2 by both Northern and Southern blot analyses (data not shown). Exposures for_glh-2_ were 14 days, using 106 dpm. This_glh-2_ signal results from use of 2-fold higher probe concentration and exposure relative to the glh-1 conditions. (F) Antisense glh-1 hybridization to whole-mount embryos. From top to bottom, the embryo stages are: 8-cell stage, >60-cell stage, and 1-cell stage. (G) Antisense glh-2 hybridization to a whole mount 1-cell embryo (Left) and a 12- to 14-cell embryo (Right). Embryos were exposed for 7 days with 106 dpms. (Bars: A–E, 50 μm;F and G, 20 μm.)
Figure 3
Western blot analysis using chicken yolk and mouse serum antibodies. Total C. elegans protein homogenate was resolved for 1100 V·h on an SDS/8% polyacrylamide gel and transferred to nylon-supported nitrocellulose membrane (Optibind-NC, Schleicher & Schuell). Strips were cut in half and incubated with anti-GLH antibodies as follows. Lanes: 1, preimmune yolk from one of the chickens immunized with GLH-1 peptide; 2, anti-GLH-1 chicken yolk; 3, preimmune yolk from one of the chickens immunized with GLH-2 peptide; 4, anti-GLH-2 yolk; 5, preimmune serum from one of the mice immunized with GLH-1 fusion protein; 6, anti-GLH-1 mouse serum. Prestained high molecular weight markers (GIBCO/BRL) of 202, 103, and 68 kDa are indicated.
Figure 4
Immunofluorescence staining of embryos with OIC1D4 and anti-GLH-1 and anti-GLH-2 antibodies. Embryos are oriented with anterior left and ventral down. (A). Embryo stained with affinity-purified chicken anti-GLH-1 antibodies (Left), mouse monoclonal antibody OIC1D4 (Center), and DAPI (Right). (B–D). Embryos stained with affinity-purified mouse anti-GLH-1 (Left), chicken anti-GLH-2 (Center), and DAPI (Right). (A and B) Two-cell embryos. P1 is in mitosis, and P granules are segregated to the posterior cortex destined for P2. (C) Seven-cell embryo. P2 is in mitosis, and P granules are segregated to the ventral region destined for P3. (D) Late-stage embryo showing P granules in Z2 and Z3. (Bar = 10 μm.)
Figure 5
Immunofluorescence staining of adult gonads using anti-GLH-1 and anti-GLH-2 antibodies. Each row shows a sample stained with affinity-purified mouse anti-GLH-1 antibodies (Left), chicken anti-GLH-2 (Center), and DAPI (Right). Gonad arms are oriented with distal left. (A) Distal gonad arm from a wild-type hermaphrodite. (B) Gonad arm from a wild-type male. Sperm present at the far right of each panel fail to stain with anti-GLH-1 and anti-GLH-2. (C) Gonad arm from a sterile hermaphrodite worm produced by a mother injected with antisense RNA to glh-1. (Bar = 10 μm.)
Similar articles
- Combinatorial RNA interference indicates GLH-4 can compensate for GLH-1; these two P granule components are critical for fertility in C. elegans.
Kuznicki KA, Smith PA, Leung-Chiu WM, Estevez AO, Scott HC, Bennett KL. Kuznicki KA, et al. Development. 2000 Jul;127(13):2907-16. doi: 10.1242/dev.127.13.2907. Development. 2000. PMID: 10851135 - glh-1, a germ-line putative RNA helicase from Caenorhabditis, has four zinc fingers.
Roussell DL, Bennett KL. Roussell DL, et al. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9300-4. doi: 10.1073/pnas.90.20.9300. Proc Natl Acad Sci U S A. 1993. PMID: 8415696 Free PMC article. - Genetic analysis of the Caenorhabditis elegans GLH family of P-granule proteins.
Spike C, Meyer N, Racen E, Orsborn A, Kirchner J, Kuznicki K, Yee C, Bennett K, Strome S. Spike C, et al. Genetics. 2008 Apr;178(4):1973-87. doi: 10.1534/genetics.107.083469. Genetics. 2008. PMID: 18430929 Free PMC article. - Specification and development of the germline in Caenorhabditis elegans.
Strome S, Garvin C, Paulsen J, Capowski E, Martin P, Beanan M. Strome S, et al. Ciba Found Symp. 1994;182:31-45; discussion 45-57. doi: 10.1002/9780470514573.ch3. Ciba Found Symp. 1994. PMID: 7835156 Review. - Connecting the Dots: Linking Caenorhabditis elegans Small RNA Pathways and Germ Granules.
Sundby AE, Molnar RI, Claycomb JM. Sundby AE, et al. Trends Cell Biol. 2021 May;31(5):387-401. doi: 10.1016/j.tcb.2020.12.012. Epub 2021 Jan 29. Trends Cell Biol. 2021. PMID: 33526340 Review.
Cited by
- Pachytene piRNAs control discrete meiotic events during spermatogenesis and restrict gene expression in space and time.
Ortega J, Wahba L, Seemann J, Chen SY, Fire AZ, Arur S. Ortega J, et al. Sci Adv. 2024 Oct 4;10(40):eadp0466. doi: 10.1126/sciadv.adp0466. Epub 2024 Oct 2. Sci Adv. 2024. PMID: 39356768 Free PMC article. - How germ granules promote germ cell fate.
Pamula MC, Lehmann R. Pamula MC, et al. Nat Rev Genet. 2024 Nov;25(11):803-821. doi: 10.1038/s41576-024-00744-8. Epub 2024 Jun 18. Nat Rev Genet. 2024. PMID: 38890558 Review. - The role of RNA-binding proteins in orchestrating germline development in Caenorhabditis elegans.
Albarqi MMY, Ryder SP. Albarqi MMY, et al. Front Cell Dev Biol. 2023 Jan 4;10:1094295. doi: 10.3389/fcell.2022.1094295. eCollection 2022. Front Cell Dev Biol. 2023. PMID: 36684428 Free PMC article. Review. - Germ Granules in Animal Oogenesis.
Dobrynin MA, Bashendjieva EO, Enukashvily NI. Dobrynin MA, et al. J Dev Biol. 2022 Oct 9;10(4):43. doi: 10.3390/jdb10040043. J Dev Biol. 2022. PMID: 36278548 Free PMC article. Review. - GLH/VASA helicases promote germ granule formation to ensure the fidelity of piRNA-mediated transcriptome surveillance.
Chen W, Brown JS, He T, Wu WS, Tu S, Weng Z, Zhang D, Lee HC. Chen W, et al. Nat Commun. 2022 Sep 9;13(1):5306. doi: 10.1038/s41467-022-32880-2. Nat Commun. 2022. PMID: 36085149 Free PMC article.
References
- Wolf N, Priess J, Hirsh D. J Embryol Exp Morphol. 1983;73:297–306. - PubMed
- Strome S, Wood W B. Cell. 1983;35:15–25. - PubMed
- Eddy E M. Int Rev Cytol. 1975;43:229–280. - PubMed
- Illmensee K, Mahowald A P. Exp Cell Res. 1976;97:127–140. - PubMed
Publication types
MeSH terms
Substances
LinkOut - more resources
Full Text Sources
Other Literature Sources
Molecular Biology Databases
Research Materials