Stein KC et al. (2022) Nature "Ageing exacerbates ribosome pausing to disrupt cotranslational proteostasis."

Frydman J, Stein KC, Morales-Polanco F, van der Lienden J, Rainbolt TK

[Nature, 2022]

Ageing is accompanied by a decline in cellular proteostasis, which underlies many age-related protein misfolding diseases^1,2. Yet, how ageing impairs proteostasis remains unclear. As nascent polypeptides represent a substantial burden on the proteostasis network³, we hypothesized that altered translational efficiency during ageing could help to drive the collapse of proteostasis. Here we show that ageing alters the kinetics of translation elongation in both Caenorhabditis elegans and Saccharomyces cerevisiae. Ribosome pausing was exacerbated at specific positions in aged yeast and worms, including polybasic stretches, leading to increased ribosome collisions known to trigger ribosome-associated quality control (RQC)^4-6. Notably, aged yeast cells exhibited impaired clearance and increased aggregation of RQC substrates, indicating that ageing overwhelms this pathway. Indeed, long-lived yeast mutants reduced age-dependent ribosome pausing, and extended lifespan correlated with greater flux through the RQC pathway. Further linking altered translation to proteostasis collapse, we found that nascent polypeptides exhibiting age-dependent ribosome pausing in C. elegans were strongly enriched among age-dependent protein aggregates. Notably, ageing increased the pausing and aggregation of many components of proteostasis, which could initiate a cycle of proteostasis collapse. We propose that increased ribosome pausing, leading to RQC overload and nascent polypeptide aggregation, critically contributes to proteostasis impairment and systemic decline during ageing.

Denver DR et al. (2010) Genome Res "Selective sweeps and parallel mutation in the adaptive recovery from deleterious mutation in Caenorhabditis elegans."

Estes S, Denver DR, Wilhelm LJ, Stein KC, Phillips PC, Palmer CA, Howe DK, Anderson JL

[Genome Res, 2010]

Deleterious mutation poses a serious threat to human health and the persistence of small populations. Although adaptive recovery from deleterious mutation has been well-characterized in prokaryotes, the evolutionary mechanisms by which multicellular eukaryotes recover from deleterious mutation remain unknown. We applied high-throughput DNA sequencing to characterize genomic divergence patterns associated with the adaptive recovery from deleterious mutation using a Caenorhabditis elegans recovery-line system. The C. elegans recovery lines were initiated from a low-fitness mutation-accumulation (MA) line progenitor and allowed to independently evolve in large populations (N 1000) for 60 generations. All lines rapidly regained levels of fitness similar to the wild-type (N2) MA line progenitor. Although there was a near-zero probability of a single mutation fixing due to genetic drift during the recovery experiment, we observed 28 fixed mutations. Cross-generational analysis showed that all mutations went from undetectable population-level frequencies to a fixed state in 10-20 generations. Many recovery-line mutations fixed at identical timepoints, suggesting that the mutations, if not beneficial, hitchhiked to fixation during selective sweep events observed in the recovery lines. No MA line mutation reversions were detected. Parallel mutation fixation was observed for two sites in two independent recovery lines. Analysis using a C. elegans interactome map revealed many predicted interactions between genes with recovery line-specific mutations and genes with previously accumulated MA line mutations. Our study suggests that recovery-line mutations identified in both coding and noncoding genomic regions might have beneficial effects associated with compensatory epistatic interactions.

Donlin MJ (2007) Curr Protoc Bioinformatics "Using the Generic Genome Browser (GBrowse)."

Donlin MJ

[Curr Protoc Bioinformatics, 2007]

A genome browser is software that allows users to visualize DNA, protein, or other sequence features within the context of a reference sequence, such as a chromosome or contig. The Generic Genome Browser (GBrowse) is an open-source browser developed as part of the Generic Model Organism Database project (Stein et al., 2002). GBrowse can be configured to display genomic sequence features for any organism and is the browser used for the model organisms Drosophila melanogaster (Grumbling and Strelets, 2006) and Caenorhabditis elegans (Schwarz et al., 2006), among others. The software package can be downloaded from the web and run on a Windows, Mac OS X, or Unix-type system. Version 1.64, as described in this protocol, was released in November 2005, but the software is under active development and new versions are released about every six months.

Donlin MJ (2009) Curr Protoc Bioinformatics "Using the Generic Genome Browser (GBrowse)."

Donlin MJ

[Curr Protoc Bioinformatics, 2009]

A genome browser is software that allows users to visualize DNA, protein, or other sequence features within the context of a reference sequence, such as a chromosome or contig. The Generic Genome Browser (GBrowse) is an open-source browser developed as part of the Generic Model Organism Database project (Stein et al., 2002). GBrowse can be configured to display genomic sequence features for any organism and is the browser used for the model organisms Drosophila melanogaster (Grumbling and Strelets, 2006) and Caenorhabditis elegans (Schwarz et al., 2006), among others. The software package can be downloaded from the Web and run on a Windows, Mac OS X, or Unix-type system. Version 1.64, as described in the original protocol, was released in November 2005, but the software is under active development and new versions are released about every six months. This update includes instructions on updating existing data sources with new files from NCBI.

Estes S et al. (2011) BMC Evol Biol "Natural variation in life history and aging phenotypes is associated with mitochondrial DNA deletion frequency in Caenorhabditis briggsae."

Smith SW, Hicks KA, Martha SR, Estes S, Denver DR, de Haan G, Knapp JB, Coleman-Hulbert AL, Stein KC

[BMC Evol Biol, 2011]

BACKGROUND: Mutations that impair mitochondrial functioning are associated with a variety of metabolic and age-related disorders. A barrier to rigorous tests of the role of mitochondrial dysfunction in aging processes has been the lack of model systems with relevant, naturally occurring mitochondrial genetic variation. Toward the goal of developing such a model system, we studied natural variation in life history, metabolic, and aging phenotypes as it relates to levels of a naturally-occurring heteroplasmic mitochondrial ND5 deletion recently discovered to segregate among wild populations of the soil nematode, Caenorhabditis briggsae. The normal product of ND5 is a central component of the mitochondrial electron transport chain and integral to cellular energy metabolism. RESULTS: We quantified significant variation among C. briggsae isolates for all phenotypes measured, only some of which was statistically associated with isolate-specific ND5 deletion frequency. We found that fecundity-related traits and pharyngeal pumping rate were strongly inversely related to ND5 deletion level and that C. briggsae isolates with high ND5 deletion levels experienced a tradeoff between early fecundity and lifespan. Conversely, oxidative stress resistance was only weakly associated with ND5 deletion level while ATP content was unrelated to deletion level. Finally, mean levels of reactive oxygen species measured in vivo showed a significant non-linear relationship with ND5 deletion level, a pattern that may be driven by among-isolate variation in antioxidant or other compensatory mechanisms. CONCLUSIONS: Our findings suggest that the ND5 deletion may adversely affect fitness and mitochondrial functioning while promoting aging in natural populations, and help to further establish this species as a useful model for explicit tests of hypotheses in aging biology and mitochondrial genetics.

Consortium Wormbase et al. (2000) West Coast Worm Meeting "From ACeDB to a more complete and usable database"

Foltz NL, Consortium Wormbase, Schwarz EM, Sternberg PW

[West Coast Worm Meeting, 2000]

We briefly describe the current status and plans for WormBase, initially an extension of the existing ACeDB database with a new user interface. The WormBase consortium includes the team that developed ACeDB (Richard Durbin and colleagues at the Sanger Centre; Jean Thierry-Mieg and colleagues at Montpellier); Lincoln Stein and colleagues at Cold Spring Harbor, who developed the current web interface for WormBase; and John Spieth and colleagues at the Genome Sequencing Center at Washington University, who along with the Sanger Centre team, continue to annotate the genomic sequence. The Caltech group will curate new data including cell function in development, behavior and physiology, gene expression at a cellular level, and gene interactions. Data will be extracted from the literature, as well as by community submission. We look forward to providing the C. elegans and broader research community easy access to vast quantities of high quality data on C. elegans. Also, we welcome your suggestions and criticism at any time. WormBase can be accessed at www.wormbase.org.

Sheldon J McKay et al. (2005) International Worm Meeting "Genome organization and colinearity in three Caenorhabditis species"

Lincoln D Stein, Sheldon J McKay

[International Worm Meeting, 2005]

C. elegans and C. briggsae are morphologically similar but their genomes have had about 100 million years to diverge. Examples of ways in which the two genomes have diverged include not only nucleotide substitutions but also species-specific expansion of gene families and many inter- and intra-chromosomal rearrangements (1). In addition to coding DNA and other functional sequences, higher order chromosome structure is also under selective constraints, for example against loss of genetic material due to non-reciprocal chromosome rearrangements. Conservation of regions of colinearity between divergent genomes suggests that gene order is also important. A striking example of this is the high degree inter-species conservation of gene order and composition of operons. We are building on the previously described comparison of the C. elegans and C. briggsae genomes using a global analysis of conservation of syntenic blocks in the genomes of these two species as well as that of C. remanei. The foundation of these comparisons is sequence similarity-based genome alignments performed by WABA(2) and blastz(3). Although conservation at the nucleotide sequence level is helpful in understanding genome evolution, global application of DNA sequence alignments in divergent genomes can be confounded by multiple rearrangements affecting the same region of the chromosome. Disruption in alignable sequences caused by a complex history of local rearrangements can mask larger blocks of colinearity that may be functionally significant. To identify such regions in these three species and, hopefully, unravel some of the complexity of nested chromosome rearrangements, we are applying a method based on the dynamic programming algorithm described in Stein et al. (1) and comparing it to the approach developed by Kent et al. (4) for the mouse and human genomes. We will discuss our findings in relation to the existing body of knowledge on C. elegans and C. briggsae genome organization and the impact of adding an additional species on our understanding of Caenorhabditis genome evolution. 1. Stein, L. D. et al., PLoS Biology 1:166-192 2. Kent, W. J. and A. M. Zahler, Genome Res 10:1115-1125 3. Schwartz, S. et al., Genome Res 13:103-107 4. Kent, W. J. et al., PNAS 100:11484-11489

Memar N et al. (2018) Dev Biol "Twenty million years of evolution: The embryogenesis of four Caenorhabditis species are indistinguishable despite extensive genome divergence."

Conradt B, Hennig C, Schiemann S, Findeis D, Memar N, Schnabel R

[Dev Biol, 2018]

The four Caenorhabditis species C. elegans, C. briggsae, C. remanei and C. brenneri show more divergence at the genomic level than humans compared to mice (Stein et al., 2003; Cutter et al., 2006; Cutter et al., 2008). However, the behavior and anatomy of these nematodes are very similar. We present a detailed analysis of the embryonic development of these species using 4D-microscopic analyses of embryos including lineage analysis, terminal differentiation patterns and bioinformatical quantifications of cell behavior. Further functional experiments support the notion that the early development of all four species depends on identical induction patterns. Based on our results, the embryonic development of all four Caenorhabditis species are nearly identical, suggesting that an apparently optimal program to construct the body plan of nematodes has been conserved for at least 20 million years. This contrasts the levels of divergence between the genomes and the protein orthologs of the Caenorhabditis species, which is comparable to the level of divergence between mouse and human. This indicates an intricate relationship between the structure of genomes and the morphology of animals.

Bhaskar Reddy Gavinolla et al. (2007) International Worm Meeting "Interactions between the microtubule severing protein MEI-2 and spindle checkpoints."

Paul E. Mains, Bhaskar Reddy Gavinolla

[International Worm Meeting, 2007]

The spindle checkpoint protein, SAN-1/MDF-3 is expressed on mitotic centromeres. During anoxia, mutants fail to arrest the cell cycle, leading to chromosome mis-segregation and reduced viability (Nystul et. al 2003). Checkpoint proteins arrest the cell cycle by inhibiting the Anaphase Promoting Complex (APC) and san-1/mdf-3 mutants suppress APC mutants (Stein et al. 2007). We have uncovered a possible link between the microtubule severing complex MEI-1/MEI-2 and the meiotic spindle checkpoint. Like SAN-1/MDF-3, MEI-1 and MEI-2 localize to chromatin and genetically interact with APC mutants. Furthermore, yeast two hybrid data (Li et al. 2004) shows that MEI-2 and SAN-1 interact physically. Both the similar expression patterns and the yeast two-hybrid binding suggested possible genetic interaction between them, so we made double mutants of san-1 and mei-2. This resulted in increased lethality, low brood sizes and spontaneous males, indications of meiotic failure. While enhancement of mei-2 spindle formation defects might be expected by the presence of a compromised spindle checkpoint, the physical interaction and colocalization might indicate a more specific role for MEI-1/MEI-2 in monitoring spindle quality.

Shakes, Diane et al. (2009) International Worm Meeting "EMB-1 is a novel protein involved in the metaphase-to-anaphase transition."

Golden, Andy, Shakes, Diane

[International Worm Meeting, 2009]

The Anaphase Promoting Complex (APC) is a multi-subunit E3 ubiquitin ligase that promotes the metaphase-to-anaphase transition during meiotic and mitotic divisions. Temperature-sensitive (ts) mutants in mat-1, mat-2, mat-3, emb-27, and emb-30 arrest as 1-cell embryos, stuck in metaphase of meiosis I. These five genes code for five subunits of the APC. The ts alleles of emb-1 have grabbed our attention because their arrest phenotype is indistinguishable from the APC mutants. Furthermore, genetic doubles constructed between emb-1(hc62) and the APC mutants cannot be maintained at the permissive temperature, a common feature of any APC double mutant. Additionally, suppressors that suppress the APC mutants (Stein et al., 2007) also suppress emb-1. What is EMB-1 you may ask? We mapped emb-1 to a tiny interval on LG III and used RNAi to phenocopy the 1-cell arrest phenotype. Rescue and sequencing confirmed that emb-1 codes for a novel protein with no known homologies outside of Caenorhabditis species. Localization studies are underway. We propose that EMB-1 is a novel subunit or regulator of the APC in C. elegans.