On the Structure, Stability and Regulation of the Ribosomal DNA in D. melanogaster

Abstract

The major loci for the large primary ribosomal RNA genes (35S ribosomal DNA (rDNA)) exist as hundreds to thousands of tandem repeats in all organisms. The rDNA accounts for over 60% of all transcriptional output and 80% of steady-state cellular RNA with only a fraction (~50%) being actively transcribed at any one time. This repetitive nature of the rDNA gives rise to an inherent instability which contributes to intra- and inter-individual sequence and copy number variation. It has been shown that targeted deletions and natural copy number variation in the rDNA can affect global gene expression and chromatin structure (Paredes and Maggert, 2009b; Paredes et al., 2011; Paredes and Maggert, 2009a). Stressors such as diet, starvation and mutations in genes that regulate rDNA can lead to increased rDNA instability and inherited copy number loss (Aldrich and Maggert, 2015). At the cellular level, rDNA Copy Number (CN) is unstable in cancer cells compared to adjacent healthy cells (Valori et al., 2020) and may underlie stochastic gains and losses of heterochromatin during development (Bughio et al., 2019; Bughio and Maggert, 2022). This loss of rDNA is therefore expected to derepress transposable elements and result in genome instability and regulatory chaos making it an ideal model for understanding epigenetic processes. Despite this, basic principles of rDNA biology remain unknown, such as the structure and organization of elements within individual cistrons, sequence information conferring tissue-specificity and the selection of active-vs-inactive copies, the mechanics underlying a secondary regulatory system called nucleolar dominance and the unusual properties of the rDNA that allow them to magnify. The highly repetitive nature of the rDNA, its existence on multiple chromosomes, and dynamic copy number variability make it10 difficult to investigate. We recently proposed that rDNA magnification is a consequence of disrupted nucleolar dominance (Kindelay and Maggert, 2023). Here, we seek to gain further mechanistic insight into the epigenetic regulation, transcriptional activity, and overall development of mutants with altered or no active rDNA. Through targeted deletions in the rDNA of the Y chromosome, we have isolated and characterized rDNA null alleles and investigated the hypothetical “lower limit” of functional rDNA for organism viability. The remaining rDNA is mostly, if not all, inserted with R1 and R2 retrotransposons; R2 elements are highly expressed during the early 1st instar stages, identified as the lethal phase. These lethal lines, along with a previously generated and characterized semi-lethal allelic series of rDNA deletions, have exhibited the presence of magnified progeny when crossed into various genetic backgrounds that destabilize heterochromatin-induced gene silencing (e.g., Su(var)3-9), affect chromosome pairing, and when in the presence of specific X chromosomes. Thus, we have identified a correlation between rDNA copy number, transcriptional activity, and nucleolar dominance as well as multiple mechanisms for the appearance of magnification that may underly and unify two longstanding observations in Drosophila: rDNA magnification and nucleolar dominance.