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The nucleolus is the most prominent membraneless compartment within the nucleus—dedicated to the metabolism of ribosomal RNA. Nucleoli are composed of hundreds of ribosomal DNA (rDNA) repeated genes that form large chromosomal clusters, whose high recombination rates can cause nucleolar dysfunction and promote genome instability. Intriguingly, the evolving architecture of eukaryotic genomes appears to have favored two strategic rDNA locations—where a single locus per chromosome is situated either near the centromere (CEN) or the telomere. Here, we deployed an innovative genome engineering approach to cut and paste to an ectopic chromosomal location—the ~1.5 mega-base rDNA locus in a single step using CRISPR technology. This “megablock” rDNA engineering was performed in a fused-karyotype strain ofSaccharomyces cerevisiae. The strategic repositioning of this locus within the megachromosome allowed experimentally mimicking and monitoring the outcome of an rDNA migratory event, in which twin rDNA loci coexist on the same chromosomal arm. We showed that the twin-rDNA yeast readily adapts, exhibiting wild-type growth and maintaining rRNA homeostasis, and that the twin loci form a single nucleolus throughout the cell cycle. Unexpectedly, the size of each rDNA array appears to depend on its position relative to theCEN, in that the locus that isCEN-distal undergoes size reduction at a higher frequency compared to theCEN-proximal counterpart. Finally, we provided molecular evidence supporting a mechanism called paralogouscis-rDNA interference, which potentially explains why placing two identical repeated arrays on the same chromosome may negatively affect their function and structural stability.
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This content will become publicly available on July 2, 2026
Phenotypic tolerance for rDNA copy number variation within the natural range of C. elegans
The genes for ribosomal RNA (rRNA) are encoded by ribosomal DNA (rDNA), whose structure is notable for being present in arrays of tens to thousands of tandemly repeated copies in eukaryotic genomes. The exact number of rDNA copies per genome is highly variable within a species, with differences between individuals measuring in potentially hundreds of copies and megabases of DNA. The extent to which natural variation in rDNA copy number impacts whole-organism phenotypes such as fitness and lifespan is poorly understood, in part due to difficulties in manipulating such large and repetitive tracts of DNA even in model organisms. Here, we used the natural resource of copy number variation in C. elegans wild isolates to generate new tools and investigated the phenotypic consequences of this variation. Specifically, we generated a panel of recombinant inbred lines (RILs) using a laboratory strain derivative with ~130 haploid rDNA copies and a wild isolate with ~417 haploid rDNA copies, one of the highest validated C. elegans rDNA copy number arrays. We find that rDNA copy number is stable in the RILs, rejecting prior hypotheses that predicted copy number instability and copy number reversion. To isolate effects of rDNA copy number on phenotype, we produced a series of near isogenic lines (NILs) with rDNA copy numbers representing the high and low end of the rDNA copy number spectrum in C. elegans wild isolates. We find no correlation between rDNA copy number and phenotypes of rRNA abundance, competitive fitness, early life fertility, lifespan, or global transcriptome under standard laboratory conditions. These findings demonstrate a remarkable ability of C. elegans to tolerate substantial variation in a locus critical to fundamental cell function. Our study provides strain resources for future investigations into the boundaries of this tolerance.
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- Award ID(s):
- 2437133
- PAR ID:
- 10647744
- Editor(s):
- SARINAY_CENIK, ELIF
- Publisher / Repository:
- PLOS Genetics
- Date Published:
- Journal Name:
- PLOS Genetics
- Volume:
- 21
- Issue:
- 7
- ISSN:
- 1553-7404
- Page Range / eLocation ID:
- e1011759
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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