Mechanisms of Nucleolar Stress in Drosophila

Louisiana State University, Baton Rouge LA

Investigators

Abstract

Ribosomes are nano-machines within our cells that read the genetic code, carried by mRNAs, to synthesize all proteins that keep our cells alive and functional. Failures in ribosome production in cells (referred to as nucleolar stress) have far-reaching effects on the well-being of cells and the organism. Importantly, not all cells are equal and various stem cells and progenitor cells show differential sensitivities to nucleolar stress: while some stem cells and progenitor cells display acute sensitivities to nucleolar stress and die by programed cell death, many other stem cells and progenitor cells seem to be resilient to nucleolar stress. The question is, why are some stem cells and progenitor cells sensitive to nucleolar stress, while others are not? This project will use the fruit fly, Drosophila melanogaster. The project tests the hypothesis that different Drosophila neuroblasts (stem cells) in the developing larval brain display differential sensitivities to nucleolar stress. Preliminary data show that “mushroom body” neuroblasts are more resilient to nucleolar stress than are most other neuroblasts. Mushroom bodies are portions of the insect brain essential for olfaction and learning in insects. The investigators induce nucleolar stress in Drosophila by depleting the ribosome biogenesis factor, a protein called Nopp140. The research determines the mechanisms of nucleolar stress as it interferes with cell viability and function, especially during neurogenesis. This is critical, as recent evidence suggests that failure in ribosome biogenesis is a contributing factor in debilitating neurological diseases. Broader impacts will introduce basic concepts in genetics and cell biology to underrepresented students in area high schools, provide an opportunity for hands-on research training for underrepresented undergraduates, and train graduate students in advanced genetics and cell biology. Various stem cells and progenitor cells show differential sensitivities toward nucleolar stress. Haplo-insufficiency mutations in mammalian treacle, a nucleolar ribosome biogenesis factor (RBF), deplete ribosomes in select embryonic neural crest cells that die by apoptosis leading to craniofacial birth defects. Most other embryonic stem cells and progenitor cells within the embryo appear resilient to this nucleolar stress. The question is, why are there differential responses to a systemic mutation in an RBF? The project will use Drosophila as a model system to answer this question. Nucleolar stress will be induced by depleting Nopp140, a nucleolar RBF structurally and functionally related to treacle. Aim 1 tests the hypothesis that mushroom body (MB) neuroblasts within the Drosophila larval brain are resilient to nucleolar stress because they stockpile RBFs by translation of maternal transcripts during a period of continued proliferation, while most other neuroblasts normally enter a period of quiescence. Aim 1A uses mosaic analyses to determine maternal contributions of RBFs to MB neuroblasts versus other neuroblasts in wild type and Nopp140-/- larval brains. Aim 1B uses co-relative light and electron microscopy (CLEM) to assess RBF and ribosome abundances in MB neuroblasts versus other neuroblasts in wild type and null Nopp140 genetic backgrounds. Aim 2 tests the mechanism that JNK is the central nucleolar stress effector in metazoans. Drosophila JNK responds to various forms of stress, and preliminary data shows that JNK is activated upon nucleolar stress in Drosophila. However, the upstream activators of JNK upon nucleolar stress remain unknown. This project identifies these activators by using RNAi to deplete known upstream JNK activators in the Nopp140 null (nucleolar stress) background, and then scores for rescued nucleolar stress phenotypes upon loss of JNK activation. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.