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Updated: Jun 26, 2026
Medium-scale Preparation of Drosophila Embryo Extracts for Proteomic Experiments
Published on: May 30, 2017
no poles encodes a predicted E3 ubiquitin ligase required for early embryonic development of Drosophila
Julie A Merkle1, Jamie L Rickmyre, Aprajita Garg
1Department of Cell and Developmental Biology, Vanderbilt University Medical Center, U-4200 MRBIII, 465 21st Avenue South, Nashville, TN 37232, USA.
Abstract:
In a screen for cell-cycle regulators, we identified a Drosophila maternal effect-lethal mutant that we named ;no poles' (nopo). Embryos from nopo females undergo mitotic arrest with barrel-shaped, acentrosomal spindles during the rapid S-M cycles of syncytial embryogenesis. We identified CG5140, which encodes a candidate RING domain-containing E3 ubiquitin ligase, as the nopo gene. A conserved residue in the RING domain is altered in our EMS-mutagenized allele of nopo, suggesting that E3 ligase activity is crucial for NOPO function. We show that mutation of a DNA checkpoint kinase, CHK2, suppresses the spindle and developmental defects of nopo-derived embryos, revealing that activation of a DNA checkpoint operational in early embryos contributes significantly to the nopo phenotype. CHK2-mediated mitotic arrest has been previously shown to occur in response to mitotic entry with DNA damage or incompletely replicated DNA. Syncytial embryos lacking NOPO exhibit a shorter interphase during cycle 11, suggesting that they may enter mitosis prior to the completion of DNA replication. We show that Bendless (BEN), an E2 ubiquitin-conjugating enzyme, interacts with NOPO in a yeast two-hybrid assay; furthermore, ben-derived embryos arrest with a nopo-like phenotype during syncytial divisions. These data support our model that an E2-E3 ubiquitination complex consisting of BEN-UEV1A (E2 heterodimer) and NOPO (E3 ligase) is required for the preservation of genomic integrity during early embryogenesis.
Insights
The Drosophila no poles (nopo) gene, encoding an E3 ubiquitin ligase, is essential for preventing mitotic arrest during early embryogenesis. Its absence triggers DNA checkpoints, leading to developmental defects, highlighting its role in maintaining genomic integrity.
Area of Science:
- Developmental Biology
- Cell Biology
- Genetics
Background:
- Cell-cycle regulators are crucial for proper embryonic development.
- Mitotic arrest and spindle defects can lead to embryonic lethality.
- Ubiquitin ligases play key roles in regulating cell-cycle progression.
Purpose of the Study:
- To identify novel cell-cycle regulators in Drosophila.
- To elucidate the function of the no poles (nopo) gene in early embryogenesis.
- To understand the molecular mechanisms underlying nopo-related mitotic defects.
Main Methods:
- Forward genetic screen in Drosophila melanogaster.
- Identification of the nopo gene (CG5140) encoding a RING domain-containing E3 ubiquitin ligase.
- Analysis of mutant phenotypes, including spindle morphology and cell-cycle progression.
- Genetic interaction studies with DNA checkpoint kinase CHK2 and E2 ubiquitin-conjugating enzyme Bendless (BEN).
- Yeast two-hybrid assay to investigate protein interactions.
Main Results:
- The maternal-effect lethal mutant no poles (nopo) exhibits mitotic arrest with acentrosomal spindles in syncytial embryos.
- The nopo gene encodes a candidate E3 ubiquitin ligase, CG5140, with essential E3 ligase activity.
- Mutation of CHK2 suppresses nopo defects, indicating DNA checkpoint activation contributes to the phenotype.
- nopo-deficient embryos show shortened interphase, suggesting premature entry into mitosis.
- Bendless (BEN), an E2 ubiquitin-conjugating enzyme, interacts with NOPO, and BEN deficiency causes similar embryonic defects.
Conclusions:
- The E3 ubiquitin ligase NOPO is essential for preventing mitotic arrest and maintaining genomic integrity during rapid syncytial divisions.
- A DNA checkpoint, likely triggered by incomplete DNA replication, contributes significantly to the nopo mutant phenotype.
- An E2-E3 ubiquitination complex involving BEN-UEV1A and NOPO is proposed to be critical for early embryonic genomic stability.
