| dc.description.abstract | Ribosomes are the micromachines which produce the materials composing the molecular-cellular complexity of organisms. Regulation of gene expression by the translational machinery provides a layer of control over the timing, location, and amount of any given gene product and its associated functions. Protein synthesis during vertebrate development is driven by a common pool of ribosomes of two distinct origins: subunits synthesized by the mother during oogenesis and stored in the egg, and subunits produced after fertilization by the embryo. In most organisms, these two are the same.
Recently, cell type-specific expression of two zebrafish ribosomal DNA (rDNA) genes was identified (Locati et al. 2017b). One rDNA variant located on chromosome 4 was found to be specifically expressed in eggs (aka maternal type), while expression of another rDNA variant located on chromosome 5 was specific to somatic cells (aka somatic type). Critically, these rRNAs vary in about 15% of their sequence. Ribosome structural heterogeneity is an appealing occurrence as it may uncover ribosome-specific functionality shaping translational control seen in development. Since variation observed between the zebrafish rRNA types is substantial, it has the potential to affect ribosome biogenesis, structure, and function at different levels. We use this system to investigate the possibility of germ cell-specific ribosome functionalization.
This thesis contains research assessing two rDNA gene variants, the transcribed rRNAs, and the two sets of ribosome subunits they compose. We separately characterize maternal and somatic ribosomes using 6 - 120 hours post-fertilization (hpf) animals. Cryo-EM structure maps of each show compositionally different, yet structurally similar assemblies. Using transgenic labeling of maternal and somatic subunits, we confirm intersubunit compatibility forms cognate and hybrid monosomes. We show primordial germ cells transcribe the somatic rDNA gene upon genome activation, and, unexpectedly, shift transcription to the maternal rDNA gene at 72 hpf. Lastly, we demonstrate maternal ribosome-enriched translation of germ cell-specific mRNA in vivo. Zebrafish germ cells maintain a majority of maternal rDNA gene products at all measured times.
Our findings solidify the chromosome 4 rDNA variant as a germ cell-specific rDNA gene. This work clarifies the structural, molecular, and cellular consequences of cell type-specific rRNA expression on ribosome heterogeneity. We indicate a germ cell-specific ribosome functionalization and frame the zebrafish dual rDNA gene variant system for future inquiry regarding ribosome biogenesis, translation control, and germ cell development. | |
