van der Linden, Alexander et al. (2017) International Worm Meeting "Global accumulation of circRNAs during aging in C. elegans."

Bauer, Matthew, Gruner, Matthew, Voda, Alexandru-Ioan, Miura, Pedro, Gruner, Hannah, Cortes-Lopez, Mariela, van der Linden, Alexander, Cooper, Daphne

[International Worm Meeting, 2017]

Circular RNAs (circRNAs) have recently been identified as a natural occurring family of wide-spread and diverse endogenous non-coding RNAs (reviewed in Cortes-Lopez and Miura, Yale J. Biol, 2016). They are remarkably stable RNA molecules mostly generated by backsplicing events from known protein-coding genes, and are abundantly expressed in many animals, from C. elegans (Ivanov et al, Cell Rep, 2015; Memczak et al, Nature 2013), to humans. Recent reports suggest functional roles for circRNAs, such as regulating transcription, binding proteins, and as microRNA sponges. CircRNAs accumulate during aging on a genome-wide level in Drosophila and mice (Westholm et al, Cell Rep, 2014; Gruner H. et al, Sci Rep, 2016), but their potential role and function in the aging process is not known. We profiled for the first time genome-wide circRNA changes during aging in C. elegans. Using total RNA-seq of whole C. elegans from L4-staged larvae, Day-1, Day-7 and Day-10 adults, we identified 1166 circRNAs including 575 novel annotations. Individual circRNAs were validated by Northern blot, qRT-PCR, and Sanger sequencing. We identified a massive and progressive accumulation of circRNAs during aging, which is independent of linear RNA changes from shared host genes. More specifically, 290 circRNAs were significantly upregulated in Day-10 compared to Day-1 adults, whereas only 6 were downregulated. This includes a >20-fold enrichment in a circRNA derived from the gene for the CREB homolog crh-1, as verified by qRT-PCR. Other genes in longevity-associated pathways produce age-accumulated circRNAs, including daf-16 and daf-2. Unlike in other species, the age-accumulation trend appears not to be confined to neuronally expressed genes. Given the progressive accumulation of circRNAs and their resistance to decay, we propose that stability in post-mitotic tissues is the main driver of the age-upregulation trend. We are currently examining whether circRNA accumulation patterns change in mutants with altered lifespan phenotypes. Taken together, our characterization of age-accumulated circRNAs lays the foundation for future investigations into the functions of circRNAs and how they might play a role in the aging process.

PLOS Pathogens Staff (2014) PLoS Pathog "Correction: Ubiquitin-Mediated Response to Microsporidia and Virus Infection in C. elegans."

PLOS Pathogens Staff

[PLoS Pathog, 2014]

The fourth author's name is spelled incorrectly. The correct name is: Tiffany L. Dunbar. The correct citation is: Bakowski MA, Desjardins CA, Smelkinson MG, Dunbar TL, Lopez-Moyado IF, et al. (2014) Ubiquitin-Mediated Response to Microsporidia and Virus Infection in C. elegans. PLoS Pathog 10(6): e1004200. doi:10.1371/journal.ppat.1004200

Palikaras K et al. (2017) Bio Protoc "In vivo Mitophagy Monitoring in Caenorhabditis elegans to Determine Mitochondrial Homeostasis."

Palikaras K, Tavernarakis N

[Bio Protoc, 2017]

Perturbation of mitochondrial function is a major hallmark of several pathological conditions and ageing, underlining the essential role of fine-tuned mitochondrial activity (Lopez-Otin et al., 2013). Mitochondrial selective autophagy, known as mitophagy, mediates the removal of dysfunctional and/or superfluous organelles, preserving cellular and organismal homeostasis (Palikaras and Tavernarakis, 2014; Pickrell and Youle, 2015; Scheibye-Knudsen et al., 2015). In this protocol, we describe a method for assessing mitophagy in the nematode Caenorhabditis elegans.

Vincent Galy et al. (2006) European Worm Meeting "MEL-28, a Novel Nuclear Envelope and Kinetochore Protein Essential for Zygotic Nuclear Envelope Assembly"

Carmen Lopez-Iglesias, Vincent Galy, Iain W. Mattaj, Cerstin Franz, Peter Askjaer

[European Worm Meeting, 2006]

Vincent Galy, Peter Askjaer, Cerstin Franz1, Carmen Lopez-Iglesias5, and Iain W. Mattaj1. The nuclear envelope (NE) of eukaryotic cells mediates nucleo-cytoplasmic transport and contributes to control of gene expression. In most eukaryotes, the NE breaks down and is then reassembled during mitosis. Assembly of nuclear pore complexes (NPCs) and the association and fusion of nuclear membranes around decondensing chromosomes are tightly coordinated processes. Here we report the identification and characterization of MEL-28, a large conserved protein essential for the assembly of a functional NE in C. elegans embryos. RNAi depletion or genetic mutation of mel-28 severely impairs nuclear morphology and leads to abnormal distribution of both integral NE proteins and NPCs. Light microscopy and transmission electron microscopy analysis demonstrate that MEL-28 localizes at NPCs during interphase, at kinetochores in early to mid mitosis then is widely distributed on chromatin late in mitosis. We show that MEL-28 is an early assembling, stable NE component required for all aspects of NE assembly. Based on its dynamic localization, we suggest that MEL-28 may link chromatin to the assembling NE.

Vargas, Elizabeth et al. (2017) International Worm Meeting "Trisomy correction occurs during both meiosis I and meiosis II."

Korf, Ian, Friedman, Jacob, McNally, Francis, McNally, Karen, Vargas, Elizabeth, Wang, David

[International Worm Meeting, 2017]

The vast majority of eukaryotes have exactly two copies of each chromosome and reproduce sexually through the process of meiosis. Accurate chromosome segregation requires the physical attachment of exactly two homologous chromosomes by a crossover so that each homologous chromosome can form attachments to opposite poles of a bipolar spindle. Thus a third copy of a chromosome might be expected to segregate randomly, resulting in 50% of progeny inheriting the extra chromosome. Hodgkin et al. (Genetics 91:67) reported that C. elegans with a third copy of the X chromosome have far fewer than 50% trisomic progeny. Cortes et al. (Elife 4:e06056) found that the preferential elimination of the extra X occurs during anaphase I of female meiosis. To test whether extra copies of all C. elegans chromosomes are preferentially eliminated, we scored the inheritance of three different DNA polymorphisms for each autosome among the progeny of triploid hermaphrodites mated with diploid males. For all 5 autosomes, the frequency of trisomic offspring was significantly less than expected from random segregation. Preferential elimination of the extra copy of chromosome V was confirmed by FISH. We also generated a trisomy IV worm strain and when trisomy IV hermaphrodites were mated to diploid males, the frequency of triplo IV progeny was again much lower than expected from random segregation. Cortes et al. (2015) found that univalent X chromosomes do not lose cohesion during anaphase I and thus lag before being preferentially captured by the polar body. Counting of mCherry:histone-labeled chromosomes in triploids revealed that chromosome number decreased between metaphase I and metaphase II and decreased again between metaphase II and diakinesis in the adult progeny. Data from live imaging of anaphase chromosome segregation and REC-8 staining of fixed meiotic spindles support a model in which some univalents remain intact during anaphase I whereas other univalents segregate apart at anaphase I. The resulting single chromatids then lag during anaphase II and are preferentially captured by the second polar body. These data demonstrate that asymmetric female meiotic divisions can correct triploidy and trisomy in C. elegans.

Schiffer, Jodie et al. (2017) International Worm Meeting "How do worms determine their readiness to cope with environmental stress?"

Stumbur, Stephanie, Heath, William, Tam, Hannah, McGowan, Natalie, Vogelaar, Abigail, Schiffer, Jodie, Apfeld, Javier, Stanley, Julian

[International Worm Meeting, 2017]

At any time in their life, worms may encounter harmful environmental conditions such as high concentrations of oxidants or high temperature. We are interested in understanding how worms prepare for these potentially lethal events. We want to know: what establishes how prepared they are? To determine the mechanisms that control survival under harmful conditions, we are examining the effects of strong loss-of-function and null mutants in a collection of intercellular signaling receptors and transcription factors regulated by these receptors. We measure survival under oxidative stress and at high temperature using a "Lifespan Machine" cluster of flat-bed scanners [1]. This automated technology presents substantial advancements in throughput and sensitivity compared to manual methods. Using this approach, we have identified several signaling receptors and transcription factors that regulate survival under oxidative conditions. Interestingly, we identified both receptors and transcription factors that function to either confer or limit oxidative-stress resistance. This indicates that the combined level of activity of these genes sets the worm's readiness to cope with oxidative stress. We are currently investigating whether these genes act together or independently to determine the worm's normal level of resistance to various stressors, and the mechanisms that establish the normal activity levels of each of these genetic determinants of stress resistance. Reference: 1. Stroustrup N, Ulmschneider BE, Nash ZM, Lopez-Moyado IF, Apfeld J, Fontana W. The Caenorhabditis elegans Lifespan Machine. Nature Methods. 2013;10(7):665-70.

Lakshmanan LN et al. (2018) Aging Cell "Clonal expansion of mitochondrial DNA deletions is a private mechanism of aging in long-lived animals."

Gruber J, Gunawan R, Yee Z, Ng LF, Lakshmanan LN, Halliwell B

[Aging Cell, 2018]

Disruption of mitochondrial metabolism and loss of mitochondrial DNA (mtDNA) integrity are widely considered as evolutionarily conserved (public) mechanisms of aging (Lopez-Otin et al., Cell, 153, 2013 and 1194). Human aging is associated with loss in skeletal muscle mass and function (Sarcopenia), contributing significantly to morbidity and mortality. Muscle aging is associated with loss of mtDNA integrity. In humans, clonally expanded mtDNA deletions colocalize with sites of fiber breakage and atrophy in skeletal muscle. mtDNA deletions may therefore play an important, possibly causal role in sarcopenia. The nematodeCaenorhabditis elegansalso exhibits age-dependent decline in mitochondrial function and a form of sarcopenia. However, it is unclear if mtDNA deletions play a role inC. elegansaging. Here, we report identification of 266 novel mtDNA deletions in aging nematodes. Analysis of the mtDNA mutation spectrum and quantification of mutation burden indicates that (a) mtDNA deletions in nematode are extremely rare, (b) there is no significant age-dependent increase in mtDNA deletions, and (c) there is little evidence for clonal expansion driving mtDNA deletion dynamics. Thus, mtDNA deletions are unlikely to drive the age-dependent functional decline commonly observed inC. elegans. Computational modeling of mtDNA dynamics inC. elegansindicates that the lifespan of short-lived animals such asC. elegansis likely too short to allow for significant clonal expansion of mtDNA deletions. Together, these findings suggest that clonal expansion of mtDNA deletions is likely a private mechanism of aging predominantly relevant in long-lived animals such as humans and rhesus monkey and possibly in rodents.

Mansfield, R. et al. (2015) International Worm Meeting "Disruption of cellular proteostasis drives sleep behavior in C. elegans."

Mansfield, R., Van Buskirk, C.

[International Worm Meeting, 2015]

Sleep is widely regarded to be a restorative state, and the physiological perturbations associated with sleep deprivation are extensive. Despite intensive study, none of these perturbations has in turn been shown to drive sleep. Recently our lab has shown that in C. elegans, environmental stressors such as heat, cold and toxins induce a sleep-like state (Hill et al., 2014). As noxious environmental stressors are known to interrupt normal proteostasis, the existence of a subsequent sleep state suggests that sleep serves to assist in the restoration of normal proteostasis. This idea is supported by our observation that sleepless animals have impaired survival following severe stress (Hill et al., 2014). Further, we have found that chaperone response defective mutants display exaggerated sleep responses. These mutants include animals lacking the stress-induced transcription factors hsf-1/HSF-1, daf-16/FOXO and a component of the endoplasmic reticulum stress-response pathway hsp-4/BiP. We are testing site-of-action for this effect by examining strains with tissue-specific rescue of HSF-1. In addition, we are using pharmacological inhibitors of the proteasome to test whether direct inhibition of the proteostasis machinery can trigger sleep. Last, we are performing RNAi against both positive and negative regulators of the heat shock transcriptional response, and screening for sleep phenotypes. The results of these efforts will be presented.Hill AJ, Mansfield R, Lopez JMG, Raizen DM, Van Buskirk C (2014) Cellular stress induces a protective sleep-like state in C. elegans. Curr Bio 24:1-7.Zimmerman JE, Naidoo N, Raizen DM, Pack AI (2008) Conservation of sleep: insights from non-mammalian model systems. Trends Neurosci 31:371-6.

Braukmann, Fabian et al. (2015) International Worm Meeting "Extracellular vesicle (ecv)RNA in C. elegans."

Braukmann, Fabian, Miska, Eric

[International Worm Meeting, 2015]

In all domains of life, non-coding RNAs regulate gene expression1. This mechanism called RNA interference has been a powerful technique in basic research over the last two decades. Today, RNAi enters the hospital and RNAi therapeutics have the potential to revolutionize drug development 2. One of the key challenges for RNAi therapeutics remains efficient RNA delivery. RNAi effectors move from cell to cell and spread through tissues in invertebrates, plants and fungi 3. In the mammalian system, extracellular vesicles (ECVs) shuttle RNA between cells 4. Recently, Wang et al. showed that C. elegans secrets ECVs in to the environment, possibly delivering RNA from animal to animal 5. However, the presents of RNA in ECVs and the molecular mechanism of mobile RNA between individuals is not known in any system. Here, I propose a purification protocol for extracellular vesicle (ecv)RNA released by the model organism C. elegans. Using this protocol, we sequenced small RNAs and RNAs from males, hermaphrodites and males deficient in vesicle release. A subset of small non-coding RNAs and messenger RNAs were enriched in male ECVs. This work characterizes the ecvRNA profile of C. elegans and may help to elucidate the social role of RNAs and has the potential to inform new approaches to RNAi therapy.1. Cech, T. R. & Steitz, J. A. The noncoding RNA revolution-trashing old rules to forge new ones. Cell 157, 77-94 (2014).2. Ozcan, G., Ozpolat, B., Coleman, R. L., Sood, A. K. & Lopez-Berestein, G. Preclinical and clinical development of siRNA-based therapeutics. Adv. Drug Deliv. Rev. (2015). doi:10.1016/j.addr.2015.01.0073. Sarkies, P. & Miska, E. A. Molecular biology. Is there social RNA? Science 341, 467-468 (2013).4. Valadi, H. et al. Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat. Cell Biol. 9, 654-659 (2007).5. Wang, J. et al. C. elegans ciliated sensory neurons release extracellular vesicles that function in animal communication. Curr. Biol. 24, 519-25 (2014).

Espinosa F et al. (2001) J Gen Physiol "Dynamic interaction of S5 and S6 during voltage-controlled gating in a potassium channel."

Joho RH, Fleischhauer R, Espinosa F, McMahon A

[J Gen Physiol, 2001]

A gain-of-function mutation in the Caenorhabditis elegans exp-2 K+-channel gene is caused by a cysteine-to-tyrosine change (C480Y) in the sixth transmembrane segment of the channel (Davis, M.W, R. Fleisch-hauer, J.A. Dent, R.H.Joho, and L. Avery. 1999. Science. 286:2501-2504). In contrast to wild-type EXP-2 channels, homotetrameric C480Y mutant channels are open even at - 160 mV, explaining the lethality of the homozygous mutant. We modeled the structure of EXP-2 on the 3-D scaffold of the K+ channel KcsA. In the C480Y mutant, tyrosine 480 protrudes from S6 to near S5, suggesting that the bulky side chain may provide steric hindrance to the rotation of S6 that has been proposed to accompany the open-closed state transitions (Perozo, E., D.M. Cortes, and L.G. Cuello. 1999. Science. 285:73-78). We tested the hypothesis that only small side chains at position 480 allow the channel to close, but that bulky side chains trap the channel in the open state. Mutants with small side chain substitutions (Gly and Ser) behave like wild type; in contrast, bulky side chain substitutions (Trp, Phe, Leu, Ile, Val, and His) generate channels that conduct K+ ions at potentials as negative as - 120 mV. The side chain at position 480 in S6 in the pore model is close to and may interact With a conserved glycine (G421) in S5. Replacement of G421 with bulky side chains also leads to channels that are trapped in an active state, suggesting that S5 and S6 interact with each other during voltage-dependent open-closed state transitions, and that bulky side chains prevent the dynamic changes necessary for permanent channel closing. Single-channel recordings show that mutant channels open frequently at negative membrane potentials indicating that they fail to reach long-lasting, i.e., stable, closed states. Our data support a "two-gate model" with a pore gate responsible for the brief, voltage-independent openings and a separately located, voltage-activated gate (Liu, Y, and R.H.Joho. 1998. Pflugers Arch. 435: 654-661).