Two differentially stable rDNA loci coexist on the same chromosome and form a single nucleolus

Area of Science:

• Genetics
• Molecular Biology
• Cell Biology

Background:

• The nucleolus, a key nuclear compartment, is responsible for ribosomal RNA (rRNA) metabolism.
• Ribosomal DNA (rDNA) clusters are prone to high recombination rates, potentially causing nucleolar dysfunction and genome instability.
• Eukaryotic genomes often position rDNA loci near centromeres (CEN) or telomeres.

Purpose of the Study:

• To investigate the impact of repositioning large rDNA loci using genome engineering.
• To mimic and monitor rDNA migratory events, creating twin rDNA loci on the same chromosome arm.
• To understand the functional and structural consequences of co-localized rDNA arrays.

Main Methods:

• Utilized CRISPR technology for "megablock" genome engineering to cut and paste a ~1.5 megabase rDNA locus.
• Engineered a fused-karyotype strain of Saccharomyces cerevisiae to create twin rDNA loci.
• Analyzed yeast adaptation, growth, rRNA homeostasis, nucleolus formation, and rDNA array size.

Main Results:

• Engineered yeast with twin rDNA loci exhibited wild-type growth and maintained rRNA homeostasis.
• Twin rDNA loci formed a single nucleolus throughout the cell cycle.
• The CEN-distal rDNA array showed a higher frequency of size reduction compared to the CEN-proximal array.
• Evidence for paralogous cis-rDNA interference was provided.

Conclusions:

• Yeast can adapt to the presence of twin rDNA loci, maintaining essential cellular functions.
• rDNA array size is influenced by its position relative to the centromere.
• Paralogous cis-rDNA interference may explain functional and stability issues in co-localized rDNA arrays.