Hall, A.N. et al. (2019) International Worm Meeting "The role of ribosomal DNA copy number in aging."

Morton, E.A., Queitsch, C., Hall, A.N.

[International Worm Meeting, 2019]

Ribosome biogenesis is known to affect lifespan. The ribosome complement of the cell requires copious amounts of ribosomal RNA, which is encoded in large repetitive ribosomal DNA (rDNA) arrays in all eukaryotes. In C. elegans, N2 has 100 copies of the rDNA repeat unit, while wild isolates have ~70-400 copies. Phenotypically, severe reductions in rDNA copy number that limit ribosome biogenesis can be fatal. However, it is poorly understood how rDNA copy number variation within the range permissible for life affects phenotype. We seek to determine if differences in rDNA copy number within the range typically found in C. elegans wild isolates affect aging. To explore the potential role of rDNA copy number in aging, we have generated a set of 118 recombinant inbred lines (RILs) with high (420 copies, strain MY1) and low (130 copies, strain SEA51) rDNA copy number. We selected individuals homozygous for either 130 or 420 rDNA copies and use an automated worm imaging system (the WormBot) to determine lifespan in high-throughput. Our data will allow us to test if rDNA copy number affects lifespan, and if rDNA copy number interacts with other genetic loci. Our data indicate that the RILs show a wide range of lifespans. We are currently exploring what genetic variation contributes to this variation in lifespan. To supplement this RIL resource, we backcrossed the 130-copy rDNA array into the background of the parental 420-copy donor, and the 420-copy array into the parental 130-copy donor. These strains allow us to isolate rDNA copy number from other genetic variation. We will use this resource to test the effect of rDNA copy number variation on healthspan traits less amenable to high-throughput testing.

Morton, E. et al. (2019) International Worm Meeting "Exploring the potential role of rDNA copy number in RNAi efficiency using a RIL population of extreme rDNA copy numbers."

Morton, E., Hall, A., Queitsch, C.

[International Worm Meeting, 2019]

Ribosomal RNAs contribute structurally and catalytically to the ribosome and make up the bulk (>80%) of the RNA content of the cell. Equally remarkable are the properties of the genes encoding these rRNAs: loci known as the rDNA. All eukaryotes maintain rDNA genes in large tandemly-repeated arrays. However, the copy number of rDNA units in the arrays varies drastically among species, strains, and individuals. Wild isolates of C. elegans have been characterized with rDNA copy number as low as 70 or as high as 400 copies per haploid genome, qualities that in our hands are stable even under laboratory propagation. We are interested in exploring if this major, largely uncharacterized, source of genomic variation contributes to phenotypic variation. Functionality of the RNA interference pathway is a promising candidate phenotype, due to observations that RNAi pathway components can localize to the nucleolus, an organelle formed around the rDNA, and that RNAi pathways have been implicated in regulation of rRNA expression. To investigate the role that rDNA copy number may have in RNAi efficiency and other phenotypes, as well as explore what genetic background differences may exist to allow such high disparity in copy number, we first characterized two strains: a GFP reporter strain, SEA51, with ~130 copies of the rDNA repeat, and MY1, a wild isolate with ~420 copies of the rDNA. We crossed these two strains to generate a resource of 118 recombinant inbred lines, approximately half of which are homozygous for the 130-copy rDNA locus and half homozygous for the 420-copy locus. We subjected these RILs to a suite of phenotyping assays, including quantification of RNAi sensitivity via growth of worms on embryonic lethal RNAi clones. We observe that the parental strains, MY1 and SEA51, differ in their sensitivity to these RNAi clones, despite the fact that neither parent has the previously-characterized ppw-1 RNAi resistance allele. The RILs exhibited large variation in their RNAi sensitivity, transgressing the parental phenotypes. RNAi sensitivity did not segregate with rDNA copy number, indicating that this locus alone does not account for the parental phenotypes, which likely have multiple contributing loci.

Mok, C.A. et al. (2017) International Worm Meeting "Repeat region inversion probes: a high-throughput estimation of rDNA locus copy number in Caenorhabditis elegans."

Mok, C.A., Queitsch, C., Waterston, R.H., Morton, E.A

[International Worm Meeting, 2017]

In C. elegans the major rDNA locus lies on the right end of LGI. A 7.2 kb repeat contains the rrn-1(18s rRNA), rrn-2(5.8s rRNA), and rrn-3(26s rRNA) gene. The N2 laboratory strain has been estimated to contain between 80-100 copies (Sulston and Brenner 1974; C. elegans sequencing consortium 1998). However, the copy number was found to vary between 55-130 copies of this region across 2007 mutagenized strains using whole genome sequencing (WGS) (Thompson et al. 2013). In contrast, wild isolates have estimated copy numbers of the LGI locus ranging as low as 68 and as high as 418 (Thompson et al. 2013). Although the standard laboratory strain N2 is usually considered to be isogenic, we wondered if recombination errors in meiosis might produce variable copy numbers of this repeat that could be associated with phenotypic differences observed in areas such as lifespan, stress response, and mutation penetrance. Two methods for estimating rDNA copy number are contour-clamped homogeneous electric field (CHEF) gels and WGS. CHEF gels, the gold standard in rDNA copy number estimation, require large numbers of worms to generate a sample. WGS, however, requires far less sample input but is costly with large sample numbers. Since examining the effect of rDNA copy number on phenotype variation requires working with small populations and multiple biological replicates, we devised a targeted sequencing approach that operates well with low sample input and is cost-efficient. Briefly, the method uses molecular inversion probes (MIPs, Hiatt et al. 2013) to sample regions of the rDNA locus as well as single-copy sites of the C. elegans genome. These probes are used in a Repeat Region Inversion Probe (RRIP) capture reaction combining genomic DNA samples with a special normalization plasmid containing single-copy versions of the rDNA locus and the other RRIP genomic targets. The plasmid versions, however, contain single nucleotide variants to distinguish these from the endogenous loci. After sequencing, reads from both plasmid and genomic DNA are analysed to generate an rDNA copy number estimate. We investigated the accuracy of the RRIP approach by testing wild isolate genomic samples that were previously sequenced by WGS in the Million Mutation Project. Our RRIP approach provides similar estimates of rDNA copy numbers with only a fraction of the reads required for WGS. Thus, RRIP is able to estimate rDNA copy number from small amounts of genomic DNA, for low sequencing cost, in a short period of time (3-day turnaround). With RRIP we can study small populations, potentially at a timescale of a single generation to investigate the influence of rDNA copy number variation in lifespan and overall health.

Morton EA et al. (2023) Genetics "Substantial rDNA copy number reductions alter timing of development and produce variable tissue-specific phenotypes in C. elegans."

Morton EA, Hall AN, Queitsch C, Cuperus JT

[Genetics, 2023]

The genes that encode ribosomal RNAs are present in several hundred copies in most eukaryotes. These vast arrays of repetitive ribosomal DNA (rDNA) have been implicated not just in ribosome biogenesis, but also aging, cancer, genome stability, and global gene expression. rDNA copy number is highly variable among and within species; this variability is thought to associate with traits relevant to human health and disease. Here we investigate the phenotypic consequences of multicellular life at the lower bounds of rDNA copy number. We use the model Caenorhabditis elegans, which has previously been found to complete embryogenesis using only maternally-provided ribosomes. We find that individuals with rDNA copy number reduced to ∼5% of wild type are capable of further development with variable penetrance. Such individuals are sterile and exhibit severe morphological defects, particularly in post-embryonically dividing tissues such as germline and vulva. Developmental completion and fertility are supported by an rDNA copy number ∼10% of wild type, with substantially delayed development. Worms with rDNA copy number reduced to ∼33% of wild type display a subtle developmental timing defect that was absent in worms with higher copy numbers. Our results support the hypothesis that rDNA requirements vary across tissues and indicate that the minimum rDNA copy number for fertile adulthood is substantially less than the lowest naturally-observed total copy number. The phenotype of individuals with severely reduced rDNA copy number is highly variable in penetrance and presentation, highlighting the need for continued investigation into the biological consequences of rDNA copy number variation.

Morton EA et al. (2019) G3 (Bethesda) "Challenges and Approaches to Genotyping Repetitive DNA."

Waterston R, Nandakumar V, Queitsch C, Brewer BJ, Morton EA, Kwan E, Stamatoyannopoulos J, Queitsch K, Mok C, Hall AN

[G3 (Bethesda), 2019]

Individuals within a species can exhibit vast variation in copy number of repetitive DNA elements. This variation may contribute to complex traits such as lifespan and disease, yet it is only infrequently considered in genotype-phenotype associations. Although the possible importance of copy number variation is widely recognized, accurate copy number quantification remains challenging. Here, we assess the technical reproducibility of several major methods for copy number estimation as they apply to the large repetitive ribosomal DNA array (rDNA). rDNA encodes the ribosomal RNAs and exists as a tandem gene array in all eukaryotes. Repeat units of rDNA are kilobases in size, often with several hundred units comprising the array, making rDNA particularly intractable to common quantification techniques. We evaluate pulsed-field gel electrophoresis, droplet digital PCR, and Nextera-based whole genome sequencing as approaches to copy number estimation, comparing techniques across model organisms and spanning wide ranges of copy numbers. Nextera-based whole genome sequencing, though commonly used in recent literature, produced high error. We explore possible causes for this error and provide recommendations for best practices in rDNA copy number estimation. We present a resource of high-confidence rDNA copy number estimates for a set of <i>S. cerevisiae</i> and <i>C. elegans</i> strains for future use. We furthermore explore the possibility for FISH-based copy number estimation, an alternative that could potentially characterize copy number on a cellular level.

Zhao Y et al. (2007) BMC Genomics "Poly-G/poly-C tracts in the genomes of Caenorhabditis."

Zhao Y, O'Neil NJ, Rose AM

[BMC Genomics, 2007]

ABSTRACT: BACKGROUND: In the genome of Caenorhabditis elegans, homopolymeric poly-G/poly-C tracts (G/C tracts) exist at high frequency and are maintained by the activity of the DOG-1 protein. The frequency and distribution of G/C tracts in the genomes of C. elegans and the related nematode, C. briggsae were analyzed to investigate possible biological roles for G/C tracts. RESULTS: In C. elegans, G/C tracts are distributed along every chromosome in a non-random pattern. Most G/C tracts are within introns or are close to genes. Analysis of SAGE data showed that G/C tracts correlate with the levels of regional gene expression in C. elegans. G/C tracts are over-represented and dispersed across all chromosomes in another Caenorhabditis species, C. briggase. However, the positions and distribution of G/C tracts in C. briggsae differ from those in C. elegans. Furthermore, the C. briggsae dog-1 ortholog CBG19723 can rescue the mutator phenotype of C. elegans dog-1 mutants. CONCLUSIONS: The abundance and genomic distribution of G/C tracts in C. elegans, the effect of G/C tracts on regional transcription levels, and the lack of positional conservation of G/C tracts in C. briggsae suggest a role for G/C tracts in chromatin structure but not in the transcriptional regulation of specific genes.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Antika TR et al. (2023) J Biol Chem "Sequence-specific targeting of C. elegans C-Ala to the D-loop of tRNAAla."

Horng JC, Hsu HL, Nazilah KR, Wang CC, Wang TL, Wang SC, Antika TR, Chuang TH, Chrestella DJ, Wang SW, Tseng YK, Pan HC

[J Biol Chem, 2023]

Alanyl-tRNA synthetase (AlaRS) retains a conserved prototype structure throughout its biology. Nevertheless, its C-terminal domain (C-Ala) is highly diverged and has been shown to play a role in either tRNA or DNA binding. Interestingly, we discovered that Caenorhabditis elegans cytoplasmic C-Ala (Ce-C-Ala_c) robustly binds both ligands. How Ce-C-Ala_c targets its cognate tRNA and whether a similar feature is conserved in its mitochondrial counterpart remain elusive. We show that the N- and C-terminal subdomains of Ce-C-Ala_c are responsible for DNA and tRNA binding, respectively. Ce-C-Ala_c specifically recognized the conserved invariant base G¹⁸ in the D-loop of tRNA^Ala through a highly conserved lysine residue, K934. Despite bearing little resemblance to other C-Ala domains, C. elegans mitochondrial C-Ala (Ce-C-Ala_m) robustly bound both tRNA^Ala and DNA and maintained targeting specificity for the D-loop of its cognate tRNA. This study uncovers the underlying mechanism of how C. elegans C-Ala specifically targets the D-loop of tRNA^Ala.

Blumenthal TE et al. (1994) Worm Breeder's Gazette "C. elegans U2AF 65."

Zorio DAR, Lea K, Blumenthal TE

[Worm Breeder's Gazette, 1994]

C. elegans U2AF65

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.