Zisoulis, Dimitrios et al. (2009) International Worm Meeting "Comprehensive identification of endogenous Argonaute-bound miRNAs and mRNA target sequences at nucleotide level resolution in C. elegans.."

Lovci, Michael, Liang, Tiffany, Wilbert, Melissa, Zisoulis, Dimitrios, Yeo, Gene, Pasquinelli, Amy

[International Worm Meeting, 2009]

MicroRNAs (miRNAs) represent a family of genomically-encoded small RNAs that post-transcriptionally regulate gene expression. miRNAs guide Argonaute-containing protein complexes to the 3'' UTR of target mRNAs resulting in degradation and/or translational repression. However, elucidation of the miRNA target recognition mechanism is challenging because miRNAs base-pair imperfectly with their target mRNAs. Furthermore, the limited availability of experimental data describing miRNA/mRNA interactions complicates the computational prediction of miRNA targets yielding results that may not be physiologically relevant. To gain further insight into sites of functional miRNA:mRNA interactions we developed an unbiased, comprehensive strategy aimed at isolating endogenous mRNA target sequences bound by the ALG-1 Argonaute protein in C. elegans. To achieve this we combined the CLIP (Cross-linking and Immunoprecipitation) technology, employing a custom anti-ALG-1 antibody, with massive parallel sequencing. Our methodology is sensitive enough to detect previously established miRNA targets with a low false positive rate. We have identified approximately 13,237 unique Argonaute-bound sites in endogenous mRNAs, corresponding to 6,643 genes, about a third of the C. elegans genome. We intend to make the map of ALG-1-bound sequences available as a UCSC Genome Browser track, providing a valuable resource for miRNA-regulated genes to the C. elegans research community. Computational analysis of the parallel sequencing data shows that: i) Argonaute-bound sites are significantly enriched at the 3'' UTR and coding exons of mRNA transcripts but depleted at 5''UTRs and introns, ii) miRNA target sites are located in conserved regions of higher structural accessibility and are enriched in CU-motifs iv) bound regions in the 3'' UTRs display a higher pairing capacity to 5'' ends of miRNAs (seed) than exonic regions v) there is a strong correlation between the presence of ALG-1 binding sites at the 3'' UTR or coding exons of a gene and its upregulated or downregulated expression, respectively, in alg-1 (-) animals, vi) genes involved in the miRNA pathway are enriched in Argonaute-binding sites. In summary, we provide a global map of ALG-1 interactions with endogenous mRNAs at nucleotide level resolution, analyze prominent characteristics of the bound sequences and limit the sequences that need to be computationally analyzed to predict targeting miRNAs.

Broughton, James P et al. (2013) International Worm Meeting "Identification of endogenous let-7 miRNA target sites by iCLIP."

Lovci, Michael T, Yeo, Gene W, Broughton, James P, Pasquinelli, Amy E

[International Worm Meeting, 2013]

A class of small RNA molecules, known as microRNAs (miRNAs), provides post-transcriptional control of gene expression through imperfect binding to target RNAs. This flexibility in binding allows a single miRNA to guide the miRNA Induced Silencing Complex (miRISC) to a range of transcripts. The core component of miRISC, an Argonaute protein, is responsible for binding the miRNA and recruiting other miRISC factors. In C. elegans, ALG-1 is the primary Argonaute responsible for miRNA function. Past approaches for identifying miRNA targets have relied on in silico methods and reporter constructs, which lack biological context. Consequently, defining endogenous targets remains an important challenge in understanding miRNA function. Recent advances in determining RNA-protein interaction sites through the use of UV crosslinking and immunoprecipitation (CLIP) methods have provided comprehensive views of Argonaute mediated miRNA targets in a variety of systems. While these studies provide evidence for miRNA regulation of a particular transcript, they do not allow for unambiguous determination of which miRNA is responsible for that regulation. This is particularly true for families of miRNAs that share identical 'seed' sequences - nucleotides 2 through 7. To address this issue, we have adapted individual-nucleotide resolution CLIP (iCLIP) for use in C. elegans and used it to identify endogenous let-7 targets. The let-7 miRNA has previously been shown to be important in regulating C. elegans development and is highly conserved throughout bilateral animals. Additionally, let-7 has been shown to interact with non-coding RNA. In let-7(n2853) animals, mature let-7 contains a point mutation in the seed sequence, which decreases the binding efficiency of mature let-7 to its target transcripts. Additionally, let-7(n2853) mutants express significantly less mature let-7. By comparing miRNA targets identified by iCLIP between wild-type and let-7(n2853) animals, we have comprehensively identified endogenous ALG-1 mediated let-7 targets.

Tang, Ngang Heok et al. (2019) International Worm Meeting "Regulation of axon regeneration by the mRNA decay factors."

Tang, Ngang Heok, Kim, Kyung Won, Yeo, Gene, Blazie, Stephen, Chisholm, Andrew, Jin, Yishi, Xu, Suhong, Yee, Brian

[International Worm Meeting, 2019]

mRNA decay factors regulate mRNA turnover by recruiting non-translating mRNAs and targeting them for translational repression, decapping and degradation, yet it remains poorly understood how mRNA decay factors function in vivo to regulate specific cellular processes. We found that mRNA decay factors form cytoplasmic puncta in C. elegans neurons and have opposing roles in axon regrowth. While decapping enzymes such as DCAP-1/2 promote axon regrowth after injury, translational repressors such as CAR-1 and CGH-1 inhibit axon regrowth in adult animals. We find that enhanced axon regeneration in a translational repressor mutant is dependent on mitochondrial functions. We show that axon injury triggers a transient mitochondrial calcium influx, which is more sustained in animals lacking translational repressors. We use genome-wide seCLIP to identify mRNA targets of translational repression in neurons, among which a regulator of mitochondrial calcium influx appears to be a direct target. Loss of translational repression leads to elevated mRNA levels for the mitochondrial calcium regulator. Moreover, the enhanced axon regrowth and sustained axonal calcium elevation observed in translational repressor mutants is dependent on mitochondrial calcium influx regulators. Our results reveal specific roles for mRNA decay regulation in neurons and a novel pathway that controls axon regeneration through mitochondrial calcium uptake.

Van Wynsberghe, Priscilla M. et al. (2013) International Worm Meeting "The period protein homolog LIN-42 negatively regulates microRNA biogenesis in C. elegans."

Homan, Kathryn, Pasquinelli, Amy E., Finnegan, Emily F., Van Wynsberghe, Priscilla M., Yeo, Gene W., Stark, Thomas J., Angelus, Evan P.

[International Worm Meeting, 2013]

MicroRNAs (miRNAs) are essential small RNAs that post-transcriptionally regulate development in C. elegans and other species. They are encoded in the genome and transcribed into primary (pri-) miRNAs that are capped and polyadenylated. Processing of a pri-miRNA by the RNase III enzyme Drosha produces the ~70 nt precursor (pre-) miRNA, which is further processed by a second RNase III enzyme, Dicer, in the cytoplasm to form the mature miRNA. In order to produce the appropriate amount of a particular miRNA in the correct location at the correct time, proper regulation of miRNA biogenesis is essential. Here we present our work that identifies the Period protein homolog LIN-42 as a new regulator of miRNA biogenesis in C. elegans. We mapped a spontaneous suppressor of the normally lethal let-7(n2853) allele to the C terminal region of lin-42. Mutations in this allele (ap201) or a second lin-42 allele (n1089) caused increased mature let-7 miRNA levels at all time points throughout the 3rd and 4th larval stages (when mature let-7 miRNA is normally expressed). Levels of pri-let-7 and a let-7 reporter, which expresses GFP from the let-7 promoter, were also increased in lin-42(n1089) worms relative to wt. These results indicate that LIN-42 normally represses pri-let-7 transcription and thus the accumulation of let-7 miRNA. This inhibition of miRNA expression by LIN-42 is not specific to let-7, as pri- and mature levels of lin-4 and mir-35 are also increased in lin-42 mutant worms and eggs, respectively. Furthermore, RNAseq analysis shows widespread increases of mature miRNAs in lin-42 mutants in both egg and 4th larval stage worms. Thus, we propose that LIN-42 is a global regulator of miRNA biogenesis. Since LIN-42 is the homolog of Drosophila and mammalian Period proteins, our results raise the possibility that these proteins may share a conserved function in transcriptionally regulating miRNA biogenesis to ultimately control rhythmic processes.

Stephanie Yonker et al. (2002) West Coast Worm Meeting "Molecular Characterization of the Dosage Compensation Gene, dpy-21"

Stephanie Yonker, Barbara J Meyer

[West Coast Worm Meeting, 2002]

Dosage compensation in Caenorhabditis elegans equalizes X-chromosome gene expression between XO males and XX hermaphrodites. DPY-21 is one of several proteins required for proper dosage compensation. dpy-21 mutations disrupt X gene expression less severely than other dosage compensation mutations and cause only 20% larval lethality compared to extensive maternal-effect lethality. Further, unlike other dosage compensation dpy mutations which affect gene expression only in XX animals, dpy-211 mutations appear to affect X-linked gene expression in XO animals. Assays of different X-linked genes show that dpy-21 mutations can cause both elevated and reduced gene expression in males.

Shi, Cheng et al. (2019) International Worm Meeting "Insulin-like peptides and the mTOR-TFEB pathway protect C. elegans hermaphrodites from Mating-induced Death."

Shi, Cheng, Murphy, Coleen

[International Worm Meeting, 2019]

C. elegans lifespan is shortened by mating, but must be delayed long enough for successful reproduction. Susceptibility to brief mating-induced death increases with age; surprisingly, this is not due to declining health, but to loss of protection upon self-sperm depletion. Self-sperm maintains expression of a DAF-2 insulin-like antagonist, INS-37, which promotes the nuclear localization of HLH-30/TFEB, a pro-longevity regulator. Mating induces the agonist INS-8, promoting HLH-30 nuclear exit and subsequent death. In opposition to the protective role of HLH-30 and DAF-16/FOXO, TOR/LET-363 and the IIS-regulated Zn-finger transcription factor PQM-1 promote seminal-fluid-induced killing. Self-sperm maintenance of nuclear HLH-30/TFEB allows hermaphrodites to resist mating-induced death, increasing the chances that mothers will survive through reproduction. The hijacking of the IIS pathway by males is combated by the mother's expression of an insulin antagonist that keeps her healthy through the activity of pro-longevity factors, as long as she has her own sperm to utilize. ** If possible, I would prefer that this abstract please be considered with the abstract submitted by Lauren N. Booth, Travis J. Maures, Robin W. Yeo, and Anne Brunet ("Self-sperm induce resistance to the detrimental effects of sexual encounters with males in hermaphroditic nematodes").

Ana Vinuela et al. (2010) Evolutionary Biology of Caenorhabditis and Other Nematodes "Transcriptional consequences of recombination in aging C. elegans"

L. Basten Snoek, Joost A.G. Riksen, Jan E. Kammenga, Ana Vinuela

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2010]

Recently, we explored genome-wide gene expression variation and regulatory loci (eQTL) in an N2 x CB4856 recombinant inbred population of developing and aging C.elegans worms (Viuela, et al., Genome Research, 2010). Here we present gene expression profiles of the two parental strains. We explored gene expression heritability and transgression as genetic parameters for the analysis of gene expression divergence in natural isolates. Moreover, we investigated the progression of those parameters with age. Preliminary results showed a strong influence of age in the transcriptional profile as well as a changed influence of the genotype in gene expression divergences in older worms. Research is ongoing to understand those results in a context of evolutionary forces affecting gene transcription divergences between two natural isolates.

Michal Levin et al. (2010) Evolutionary Biology of Caenorhabditis and Other Nematodes "Transcriptomics on a phylogeny: comparative gene expression in the Caenorhabditis embryo"

Michal Levin, Florian Wagner, Tamar Hashimshony

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2010]

Metazoan development is a highly conserved process governed by gene regulatory circuits comprised of similar genes across species, with precise temporal and spatial gene regulation. Comparing developmental regulatory programs of both phenotypically similar and divergent organisms is a powerful test of the hypothesis that morphological evolution is predominantly based upon modulation of gene expression across time and space. Further it can provide insights into the plasticity of the genomic program for encoding phenotypically similar organisms with different underlying programs. We are exploring embryonic gene expression programs of a stable set of orthologs and quickly evolving paralogs across a phylogeny of five completely sequenced Caenorhabditis species (C.elegans, C.briggsae, C.remanei, C.brenneri and C.japonica). Custom-designed microarrays containing the entire gene set of each of the species were used to obtain expression levels of all genes across ten embryonic stages of development (4-cell stage to first larvae) of precisely staged embryos. Analysis of this data set has enabled the screening for gene expression conservation and divergence across gene families. Further, the consensus of orthologous gene expression profiles appear to be more correlated with the timings of known functions in C. elegans than the actual C. elegans gene expression profile, suggesting that gene expression profiles across species can be analyzed like gene sequences and queried for the signature of selection through observed patterns of conservations and divergences. We propose that genetic drift of the transcriptome is an important developmental principle with implications to other principles such as robustness and modularity.

Okano, A. et al. (2011) International Worm Meeting "Estimation of gene regulatory networks of C.elegans early embryo."

Nakayama, S., Monobe, K., Maeshiro, T., Ito, M., Okano, A.

[International Worm Meeting, 2011]

We have constructed a series of gene regulatory networks from microarray data of C.elegans early embryo, measured every ten minutes for initial 50 minutes starting from the single cell stage. Generated gene networks correspond to each measurement, i.e. 0, 10, 20 minutes and so on. Genome wide microarrays from Affymetrix were used. The generation method of gene regulatory network is based on the gene regulatory network of Sea urchin early embryo, which is experimentally verified network and thus of high confidence. Then genes belonging to the Sea urchin network is used as query sequence to search in C.elegans gene set. Highly homologous worm genes with significant expression value obtained from microarray data are included to the predicted gene regulatory network. Gene regulatory relationship is created if the Sea urhchin query genes are in regulatory relationship and if the expression values of C.elegans genes are not ambiguous from the gene regulatory types (activation or repression). Five gene regulatory networks were created. Network analysis is used to extract hub genes, the genes that regulate large number of other genes. The molecular function of hub genes identified based on wormbase database are: cell division, transcription factor, DNA binding, nucleotide binding, zinc ion binding, double helix binding, and enhancer binding. Temporal expression data was used for clustering process, resulting in 119 expression groups, which correspond to gene regulation groups.

Holly Sassi et al. (2005) International Worm Meeting "Gene CATCHR Gene Cloning and Tagging for C. elegans using yeast Homologous Recombination: An innovative approach for the analysis of gene expression"

Ramona Cooperstock, Holly Sassi, Stephanie Renihan, Andrew Spence

[International Worm Meeting, 2005]

To facilitate a systematic investigation of C. elegans expression patterns and interactions, we have developed Gene CATCHR (Gene Cloning and Tagging for C. elegans using yeast Homologous Recombination). Gene CATCHR is a novel method of generating reporter constructs that exploits yeast homologous recombination to subclone and tag worm genes. The cloning and tagging steps require PCR, two yeast transformations, and several platings on selective media. Each step is relatively straightforward, and amenable to high throughput techniques. Using Gene CATCHR we can produce functional tagged transgenes that provide accurate information about the spatiotemporal pattern of gene expression and subcellular localization of gene products. Major advantages of Gene CATCHR include:1) Clone size potential: we can capture large genomic regions facilitating the isolation of regulatory sequences that may be important for gene expression. We have used Gene CATCHR to clone 5 operons, up to 20 kb in size. Notably, we have successfully cloned and tagged a 36 kb genomic fragment containing the tra-1 gene.2) Flexibility: any tag can be inserted at any position in a gene without introducing extraneous sequence. Our system can also be exploited to create targeted mutations.3) Scalability: we can clone and tag many genes at once in 96 well format. To date, we have cloned 68 genes, 22 of which have been tagged with GFP. In order to generate a high throughput system, we are currently developing a bioinformatics tool.4) Expression and Function: The expression patterns generated from Gene CATCHR derived transgenes are consistent with those previously reported, and we have documented additional uncharacterized expression patterns. Our transgenes are functional in rescue assays; as proof of principle, we have rescued 5 mutants.All Gene CATCHR related protocols and reagents will be made available to the worm community through our website, www.genecatchr.org (stay tuned!). The expression data generated will also be submitted to WormBase. Gene CATCHR represents an efficient, highly versatile, and powerful system for functional genomic applications.