Carey, James F. et al. (2009) International Worm Meeting "New Roles for Mitotic Condensin Proteins in RNAi and Chromatin-Based Gene Silencing."

Hagstrom, Kirsten A., Carey, James F., Thompson, James, Yates III, John R.

[International Worm Meeting, 2009]

Genome integrity requires proper chromosome structure and faithful segregation of chromosomes during mitosis. Protein complexes called condensins are conserved key regulators of chromosome architecture, and are essential for chromosome segregation during cell division. Here we describe new roles for condensins in RNAi and chromatin-based gene regulation. Immunoprecipitation of condensin II recovered proteins that influence nucleosome positioning or histone tail modification, as well as proteins in small RNA pathways. In several functional assays, we demonstrate that C. elegans condensin complexes are involved in chromatin-based gene silencing phenomena that utilize RNAi machinery. For example, condensins are required to silence transgenes that undergo chromatin- and RNAi-dependent transcriptional gene silencing. Notably, condensin II also appears to be required for RNAi. Condensin II mutants are less sensitive to dsRNA treatment, and fail to elicit an effective RNAi response. Moreover, this effect even was observed in post-mitotic cells, suggesting a novel non-mitotic role for condensins in RNAi mediated silencing. Thus, we speculate that condensins influence a transcriptional silencing component of RNAi that has not been well characterized in worms. These findings reveal an unexpected link between condensins and gene silencing mediated through small RNA and chromatin modification, providing evidence for additional mechanisms by which condensins maintain genome integrity.

Doroquez, David B. et al. (2011) International Worm Meeting "The Role of Intracellular Trafficking in Modulating Ciliary Structure and Function in C. elegans."

Sarkeshik, Ali, Doroquez, David B., Sengupta, Piali, Yates III, John R., Olivier-Mason, Anique

[International Worm Meeting, 2011]

Primary cilia are organelles that serve as environmental sensors, and are present on most cell types in vertebrates. The structure and biogenesis of these organelles are conserved from algae to humans, allowing for parallel studies of cilia in many model systems. Defects in cilia are implicated in multiple sensory pathologies. C. elegans is an ideal system to study cilia with its tractability and the ability to analyze its 60 ciliated sensory neurons at high resolution. Individual chemosensory neurons exhibit highly specialized cilia structures that are essential for their sensory properties. Cell-specific mechanisms of intraflagellar transport (IFT) and sensory signaling contribute to ciliary structural and morphological diversity. In particular, sensory signaling is required to modulate the specialized architecture of AWB olfactory cilia, and this modulation is dependent on vesicular trafficking. Defects in trafficking are likely to affect cilia structure/function due to altered transport and localization of ciliary signaling molecules resulting in defective cellular homeostasis. The goal of this project is to study the role of intracellular trafficking in the generation and maintenance of cilia morphology. In order to identify components involved in cilia biology, we identified proteins associated with different IFT components via mass spectrometry and proteomics. Many predicted IFT-associated components were identified in this analysis, suggesting that this approach may allow us to identify new cilia-related components. We have identified trafficking proteins associated with IFT complex proteins. In particular, we identified the GTPase dynamin DYN-1 and localized it to the base of AWB cilia. Conditional dyn-1 mutants have Y-shaped cilia branches with increased membranous areas, similar in phenotype to sensory signaling mutants. Knockdown of DYN-1 produces a bulbous AWB ciliary phenotype similar to that seen in clathrin AP2 adaptor dpy-23 mutants. We also identified exocyst complex proteins in our IP-MS studies. Interestingly, we find that SEC-8 localizes to the base of AWB cilia. Our data suggest a role for endocytosis and exocytosis in contributing to ciliary function. Ongoing experiments are examining whether sensory signaling regulates rates of dendritic trafficking, exocytosis and endocytosis to maintain AWB ciliary structure. These experiments will elucidate the role of vesicular transport in regulating cilia structure and how the sculpting of specialized sensory ciliary architecture in an activity-dependent manner allows animals to respond to environmental cues.

Yuen-Yi Tseng et al. (2007) International Worm Meeting "Caenorhabditis elegans expresses a functional ArsA ATPase."

Vivian Liao, Chan-Wei Yu, Jui-Tung Liu, Yuen-Yi Tseng

[International Worm Meeting, 2007]

As arsenic is the most prevalent environmental toxin, it is imperative for us to understand the mechanisms of metalloid detoxification. In prokaryotes, arsenic detoxification is accomplished by chromosomal and plasmid-borne operon-encoded efflux systems. The bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenite (As(III)) and antimonite (Sb(III)). Herein, we describe the identification of a Caenorhabditis elegans homologue (asna-1) that encodes the ATPase component of the Escherichia coli As(III) and Sb(III) transporter. We have evaluated the responses of wild-type and asna-1 mutant nematodes to various metal ions and found that asna-1 mutant nematodes are more sensitive to As(III) and Sb(III) toxicity than that of wild-type animals. These results provide evidence that ASNA-1 is required for C. elegans'' defense against As(III) and Sb(III) toxicity. A purified maltose-binding protein (MBP)- ASNA-1 fusion protein was biochemically characterized, and its properties were compared with those of ArsAs. ATPase activity of the ASNA-1 protein was dependent on the presence of As(III) or Sb(III). As(III) stimulated ATPase activity by 2 ± 0.2 (SEM) fold, while Sb(III) stimulated it by 4.6 ± 0.15 (SEM) fold. The results indicate that As(III)- and Sb(III)-stimulated ArsA ATPase activities are not restricted to bacteria but extend to animals by demonstrating that the asna-1 gene of the nematode, C. elegans, encodes a functional ArsA ATPase whose activity is stimulated by As(III) and Sb(III) and which is critical for As(III) and Sb(III) tolerance in the intact organism.

Alessi, Amelia F. et al. (2013) International Worm Meeting "CEY-1 attenuates let-7 microRNA-mediated silencing in C. elegans."

Chun, Sang Young, Alessi, Amelia F., Khivansara, Vishal, Yates III, John R., Kim, John, Moresco, James J.

[International Worm Meeting, 2013]

MicroRNAs (miRNAs) are an abundant superfamily of small regulatory RNAs that control diverse cellular and developmental processes. MiRNAs function in the miRNA induced silencing complex (miRISC) to trigger translational repression and destabilization of target mRNAs. The miRISC is guided to targets by its bound miRNA, which recognizes partially complementary sequences in target 3'UTRs. Recent studies suggest miRISC activity is modulated by accessory factors. We have identified the RNA binding protein (RBP) CEY-1 as a negative regulator of let-7 miRISC activity. let-7 is a broadly conserved miRNA that regulates cellular differentiation in animals. Its dysregulation is also a hallmark of many cancers. C. elegans let-7 is essential for viability; it controls developmental timing, including the temporal specification of cell fate. cey-1 mutants potently suppress the hypodermal seam cell hyperplasia and highly penetrant vulval defects of let-7 hypomorphic mutants. At the molecular level, our data suggest CEY-1 binds RNA to antagonize let-7 miRISC activity: loss of cey-1 in let-7 mutants suppresses upregulation of key let-7 targets including lin-41, immunopurification of CEY-1 complexes recovers lin-41 transcripts, and high-throughput sequencing of RNA isolated by crosslinking and immunoprecipitation (HITS-CLIP) identifies CEY-1 binding sites in the 3'UTR of let-7 targets. Interestingly, CEY-1 and the let-7 biogenesis and stability factor LIN-28 are both part of a deeply conserved family of RBPs homologous to prokaryotic cold-shock proteins (CSPs). Our data implicate a novel, non-redundant function of a second CSP-like protein in modulating let-7 miRNA activity. We are currently working to further define the molecular relationship between CEY-1 and let-7 miRISC and elucidate if this relationship is functionally conserved in mammals.

Tsai, Hsin-Yue et al. (2009) International Worm Meeting "RDE-8 encodes a novel protein with a conserved domain required for RNAi."

Yates III, John R., Gu, Weifeng, Mello, Craig, Moresco, James J., Chen, Chun-Chieh G., Tsai, Hsin-Yue

[International Worm Meeting, 2009]

Several classes of small RNAs have been identified in mammals, zebrafish, Drosophila, and the nematode C. elegans. These include Dicer-dependent products (primary siRNAs and miRNAs) as well as secondary siRNAs produced by RNA-dependent RNA polymerase (RdRP). We have recently identified a new RNAi pathway component, RDE-8, which is essential for the accumulation of RdRP-derived small RNAs in both the exo- and endo- RNAi pathways. RDE-8 contains a novel protein motif that is highly conserved throughout all three kingdoms of life. Interestingly, in other organisms the majority of proteins containing the RDE-8 motif also contain RNA binding domains, most often of the CCCH zinc finger or KH domain families. Although RDE-8 itself is not highly conserved, one C. elegans RDE-8 family member (C30F12.1) has a human homolog in which both the amino acid composition and organization of its CCCH and RDE-8 motifs are well conserved. A third C. elegans RDE-8-motif protein also contains a Cytidine deaminase motif and is required for male fertility (See abstract by Colin Conine). These findings suggest that proteins containing the RDE-8 motif function in RNA binding and modification. To explore the role of RDE-8 in small-RNA pathways, we have utilized mass-spectrometry-based proteomics to identify protein interactors, and small-RNA deep sequencing to identify endogenous small RNAs whose biogenesis/stability depend on RDE-8. Our data suggests that RDE-8 is required for several distinct small-RNA pathways and resides in at least two protein complexes. RDE-8 interacts with the sap-domain exonuclease ERI-1b, and is required for the accumulation of ERI-pathway small RNAs. Interestingly, RDE-8 not only co-purifies with ERI-1b, but is required for a ssRNA trimming activity associated with the native ERI-1b/RDE-8 complex. We speculate that this trimming activity is necessary for the processing of substrates (or products) of the RdRP RRF-3. RDE-8 appears to form a second complex with components of the RNAi and transposon silencing small RNA pathway, including RDE-3, MUT-15, and MUT-16. We have not yet identified specific biochemical activities associated with this complex. However, it may also be involved in recruiting RdRPs: in this case, RRF-1 and EGO-1, which function to amplify silencing signals in the exo- and endo-RNAi pathways. We are currently investigating the biochemical properties of the RDE-8 complexes, and are exploring the activity of the RDE-8 motif.

Karishma Sadikot et al. (2006) Development & Evolution Meeting "A Novel Dosage Compensation Protein with Chromosome Segregation Functions"

Karishma Sadikot, Barbara Meyer, Nizar Taki, Gyorgyi Csankovszki, Emily Petty

[Development & Evolution Meeting, 2006]

The C. elegans dosage compensation complex (DCC) assembles on hermaphrodite X chromosomes to downregulate gene expression two-fold. This ensures equal amounts of X-linked gene products in XX hermaphrodites and XO males. The DCC is similar to the 13S condensin complex, which is essential for mitotic and meiotic chromosome segregation. The four known DCC subunits are homologous to four of the five condensin proteins. In search of the fifth DCC subunit, antibodies against the unique dosage compensation protein, DPY-27, were used to immunoprecipitate the DCC from embryonic extracts. In collaboration with Ian McLeod and John R. Yates III, MUD-PIT analyses of precipitated proteins identified a novel DCC subunit, the protein encoded by F29D11.2. PSI -BLAST identified F29D11.2 as the homolog of the condensin subunit CAP-G. Using RNAi, we confirmed that this protein plays a role in dosage compensation. F29D11.2 is required for localization of other DCC subunits, and it will only localizes to the X in the presence of all other subunits, indicating that the protein is an integral member of the worm DCC. Interestingly, F29D11.2 appears to also interact with the worm mitotic condensin. Our preliminary results indicate that the protein is present in immunoprecipitated mitotic condensin complexes. The protein also appears to localize to germline nuclei. Furthermore, animals homozygous for a null mutation in F29D11.2 arrest during larval development with chromosome segregation defects, including chromatin bridges connecting interphase nuclei. These results suggest that F29D11.2 may function in both dosage compensation and mitotic and meiotic chromosome segregation.

Maduro MF et al. (1996) West Coast Worm Meeting "THE LG III DEFICIENCY eDf2 AND ASSOCIATED FREE DUPLICATION eDp6 ARE NOT MUTUALLY COMPLEMENTARY"

Maduro MF, Pilgrim DB

[West Coast Worm Meeting, 1996]

The deficiency eDf2 and the free duplication eDp6 were obtained concomitantly following acetaldehyde mutagenesis (Hodgkin, 1980). These two aberrations have been useful in mapping many LG III loci, which have demonstrated that eDp6 contains the right third of LG III and eDf2 contains the left portion. We used the eDf2 and eDp6 breakpoints as physical markers to clone unc-119, and showed that the genomic region corresponding to the unc-119 cDNA is completely absent. The gene unc-119 is the only LG III locus to not map to either eDf2 or eDp6. Our interest in these aberrations was renewed when we ran across a report by Starich et al. on the isolation of dyf-2 which maps to the far end of LG III(R) and is complemented by both eDf2 and eDp6. Using a plasmid mini-library approach, we walked across the eDf2 breakpoint, and probed the YAC grid to find that eDf2 contains sequences from the far right end of III. What is strange is that these same sequences are also present on eDp6, suggesting that eDf2 and eDp6 do not result from a simple chromosomal break, and the use of these rearrangements for mapping genes on LG III(R) should be approached with caution. The mapping evidence will be summarized and a model for the creation of eDf2 and eDp6 will be given.

Wu, Tammy F. et al. (2009) International Worm Meeting "Global analysis of histone subtype composition in C. elegans sperm using MudPIT mass spectrometric analysis."

Yee, Michael C., Chu, Diana S., Hervold, Kieran, Wong, Catherine, Wu, Tammy F., Yates III, John R., Fitzpatrick, Colin F., Go, Aiza C.

[International Worm Meeting, 2009]

Sperm are remarkable cells specialized for the delivery of the paternal genome to the oocyte, while embryos consist of rapidly dividing, largely undifferentiated cells. Unlike embryos, sperm have jettisoned most cellular components, have highly compacted chromatin, and are thought to be largely transcriptionally inactive. Thus, sperm rely on substitution histones and post-translational modification (PTM) of existing proteins for development and function. In other organisms, replacement of S-phase histones by histone variants and protamines fundamentally changes chromosome architecture at the nucleosome level. How chromatin differs between these two tissue types has been largely unexplored. To investigate the nature of C. elegans sperm chromatin composition, we conducted a global analysis of sperm and embryo chromatin proteins using Multidimensional Protein Identification Technology (MudPIT) mass spectroscopy. One story that emerges from these data reveals that variant histone H2A proteins are incorporated to a higher degree in a terminally differentiated cell type compared to actively dividing cells. In embryos, spectral counts of peptides corresponding to the four histone H2A variants confirms S-phase histone H2A as the most abundant form, while HTZ-1 and HIS-35 (an alternative H2A that differs from S-phase H2A by one residue) are much less abundant. However, this profile differs in sperm. Here, we detect the sperm-specific incorporation of the HTAS-1 variant. HTAS-1 is required for optimal male fertility, is expressed only in sperm as detected by Western Blot and immunostaining, and intriguingly marks paternally-contributed chromatin after fertilization. Also, despite the high level of sequence identity, HIS-35 is employed in greater proportion in sperm. Interestingly, unlike S-phase H2A histone genes, his-35 contains an intron. This feature may reflect a mechanism for regulating HIS-35 expression to fine-tune chromatin status during spermatogenesis. Our novel approach to determine chromatin composition between disparate cell types has identified over 1000 proteins from embryos and sperm each, laying the groundwork for analysis of other histone subtypes as well as protamine orthologs. In addition, our ongoing large-scale identification of histone PTMs is a valuable resource to examine how paternal epigenetic marks contribute to embryonic development.

Ikegami, Kohta et al. (2013) International Worm Meeting "Integral nuclear pore components associate with Pol III-transcribed genes and are required for Pol III transcript processing in C. elegans."

Lieb, Jason, Ikegami, Kohta

[International Worm Meeting, 2013]

Nuclear pores associate with active protein-coding genes in yeast and have been implicated in transcriptional regulation. Here, we show that in addition to transcriptional regulation, key components of C. elegans nuclear pores are required for processing of small non-coding RNAs transcribed by RNA Polymerase III (Pol III). Chromatin immunoprecipitation of NPP-13 and NPP-3, two integral components of the nuclear pore, and importin-b IMB-1, provides strong evidence that this requirement is direct. All three proteins associate specifically with tRNA and small nucleolar RNA (snoRNA) genes undergoing Pol III transcription. These pore components bind immediately downstream of the Pol III pre-initiation complex, but are not required for its recruitment. Instead, NPP-13 are required for the cleavage of snoRNA precursor transcripts into mature snoRNAs, whereas processing of Pol II transcripts occurs normally. Our data suggest that integral nuclear pore proteins act to coordinate transcription and Pol III transcript processing in C. elegans.

Chan-Wei Yu et al. (2007) International Worm Meeting "Characterization and regulation of the C. elegans GCS-1 by arsenite."

Chan-Wei Yu, Vivian Liao

[International Worm Meeting, 2007]

Arsenic is a potent toxin and carcinogen. We previously demonstrated that GCS-1 is essential for the synthesis of intracellular GSH in C. elegans and consequently that the intracellular GSH status plays a critical role in protection of C. elegans from arsenic-induced oxidative stress (Yu and Liao, 2005). Herein we investigate the regulation of GCS-1 in response to As(III). Our results showed that As(III) caused a concentration-dependent increase in the GSH levels up to a concentration of 10 <font face=symbol>m</font>M. Real-time RT-PCR analysis shows that gcs-1 mRNA transcript is most abundant in L1, L2 and adult stages. Whereas GCS-1 mRNA at L1 and dauer stages exhibited higher induction ratio after 1 h of As(III) exposure. Moreover, we observed that the increase in GCS-1 mRNA expression level caused by As(III) shows dose-dependence fashion, in parallel with the increase in the GSH level. RNAi feeding assay showed that worms that were fed GCS-1 dsRNA and were grown in the presence of 0.25 mM As(III) were obviously small and development arrest. Transcription of gcs-1 was observed in the pharynx, ASI chemosensory neurons, and intestine of post-embryonic C. elegans and upon exposure to As(III), As(V), or Sb(III), the intestinal expression increases dramatically; whereas Cd(II) induced significant expression in the pharynx. Together, our results indicated that GCS-1 is developmentally regulated and it plays a critical role for the protection of C. elegans against arsenic-induced oxidative stress.