Weiner A (2003) Mol Cell "Soaking up RNAi."

Weiner A

[Mol Cell, 2003]

RNA interference (RNAi) describes the ability of double-stranded RNA (dsRNA) to inhibit homologous gene expression at the RNA or DNA level. In a recent paper, Feinberg and Hunter report that a single transmembrane dsRNA transport protein may enable RNAi to spread systemically from one cell to another.

Michael A Beer (2005) International Worm Meeting "Systematic genome-wide identification of transcriptional cis-regulatory logic in C. elegans."

Michael A Beer

[International Worm Meeting, 2005]

We have developed a systematic approach for inferring cis-regulatory logic from whole-genome microarray expression data.[1] This approach identifies local DNA sequence elements and the combinatorial and positional constraints that determine their context-dependent role in transcriptional regulation. We use a Bayesian probabilistic framework that relates general DNA sequence features to mRNA expression patterns. By breaking the expression data into training and test sets of genes, we are able to evaluate the predictive accuracy of our inferred Bayesian network. Applied to S. cerevisiae, our inferred combinatorial regulatory rules correctly predict expression patterns for most of the genes. Applied to microarray data from C. elegans[2], we identify novel regulatory elements and combinatorial rules that control the phased temporal expression of transcription factors, histones, and germline specific genes during embryonic and larval development. While many of the DNA elements we find in S. cerevisiae are known transcription factor binding sites, the vast majority of the DNA elements we find in C. elegans and the inferred regulatory rules are novel, and provide focused mechanistic hypotheses for experimental validation. Successful DNA element detection is a limiting factor in our ability to infer predictive combinatorial rules, and the larger regulatory regions in C. elegans make this more challenging than in yeast. Here we extend our previous algorithm to explicitly use conservation of regulatory regions in C. briggsae to focus the search for DNA elements. In addition, we expand the range of regulatory programs we identify by applying to more diverse microarray datasets.[3] 1. Beer MA and Tavazoie S. Cell 117, 185-198 (2004). 2. Baugh LR, Hill AA, Slonim DK, Brown EL, and Hunter, CP. Development 130, 889-900 (2003); Hill AA, Hunter CP, Tsung BT, Tucker-Kellogg G, and Brown EL. Science 290, 809812 (2000). 3. Baugh LR, Hill AA, Claggett JM, Hill-Harfe K, Wen JC, Slonim DK, Brown EL, and Hunter, CP. Development 132, 1843-1854 (2005); Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, and Kenyon C. Nature 424 277-283 (2003); Reinke V, Smith HE, Nance J, Wang J, Van Doren C, Begley R, Jones SJ, Davis EB, Scherer S, Ward S, and Kim SK. Mol Cell 6 605-616 (2000).

Lee M-H et al. (1997) International C. elegans Meeting "TOWARD THE IDENTIFICATION OF IN VIVO RNA TARGETS OF GLD-1."

Lee M-H, Schedl TB

[International C. elegans Meeting, 1997]

gld-1 is a female germ cell specific tumor suppressor gene that is essential for normal oocyte differentiation and meiotic prophase progression (Francis et al., 1995a,b). GLD-1 is a member of a small family of proteins, including the mouse/human Sam68 and Quaking proteins, that are highly related over an ~ 200 amino acid region which contains a 70 to 100 amino acid RNA binding domain called the KH motif (Jones and Schedl, 1995). Extensive mutational analysis of gld-1 has demonstrated the importance of conserved sequences for its in vivo function. GLD-1 is a cytoplasmic protein and therefore is likely to control mRNA translation or RNA stability in the cytoplasm (Jones et al., 1996). Currently, no obvious RNA targets have been identified from genetic analysis. We have employed a biochemical approach to identify in vivo RNA targets of GLD-1. The strategy is based on the ability of anti GLD-1 antibodies to immunoprecipitate (IP) GLD-1 from a worm lysate and the strong likelihood that GLD-1 present in the lysate is functional and bound to RNAs. GLD-1 was IPed with affinity purified polyclonal antibodies from a cytosol extract of adult hermaphrodites or with rabbit IgG as a control. RNAs co-IP with GLD-1, as well as with rabbit IgG, were converted into cDNAs. Non-specifically trapped RNAs from the GLD-1 IP were eliminated by subtracting cDNAs from the GLD-1 IP with cDNAs from the control IP. The difference product after 4 rounds of subtraction (DP4) was used to screen a Lambda ZAP II cDNA library constructed with the cDNAs from the GLD-1 IP. Duplicate filters were also screened with a probe made from control IP cDNA. Clones that are positive only with the DP4 probe are currently being characterized. Francis et al., 1995a Genetics 139, 579-606; Francis et al., 1995b Genetics 139, 607-630; Jones and Schedl, 1995 Genes Dev. 9, 1491-1504; Jones et al., 1996 Dev. Biol. 180, 165-183. This work was supported by NIH Grant HD25614.

Michael C Fitzgerald et al. (2004) East Coast Worm Meeting "Specificity of the C. elegans Putative Transmembrane Channel SID-1"

Craig P Hunter, Michael C Fitzgerald

[East Coast Worm Meeting, 2004]

SID-1 was identified in a genetic screen for mutants capable of cell autonomous RNAi but deficient for systemic RNAi (Winston et al., 2002). The systemic RNAi defect is likely due to the inability of cells lacking SID-1 to import double-stranded RNA (dsRNA) from neighboring cells. The nature of SID-1 activity was elucidated using a heterologous system whereby either wild-type SID-1 or, as a negative control, a missense mutant form of SID-1 was transiently expressed in Drosophila S2 cells (Feinberg & Hunter 2003). These investigations showed that SID-1 enables efficient RNAi by adding dsRNA to the media of transfected S2 cells (soaking RNAi) and uptake of labeled dsRNA into cells. Furthermore, long dsRNA was shown to be more effective than short dsRNA for SID-1 mediated soaking RNAi in S2 cells and for systemic RNAi in C. elegans . We are investigating the length dependence of silencing as well as substrate specificity of the SID-1 channel. We have shown that SID-1 is extremely efficient, enabling soaking RNAi with less than one molecule of dsRNA per transfected cell and will present evidence that transport is extremely rapid. We will also report the results of ongoing analyses of length-dependent transport and channel selectivity, which may have implications for the use of SID-1 as a tool for molecular biology. W. M. Winston, C. Molodowitch, C. P. Hunter, Science 295 , 2456-59 (2002). Systemic RNAi in C. elegans requires the putative transmembrane protein SID-1. E. H. Feinberg, C. P. Hunter, Science 301 , 1545-7 (2003). Transport of dsRNA into cells by the transmembrane protein SID-1.

Julia M Claggett et al. (2004) East Coast Worm Meeting "An Investigation into the potential regulatory relationships between components of a network specified by pal-1."

L Ryan Baugh, Julia M Claggett, Craig P Hunter

[East Coast Worm Meeting, 2004]

PAL-1 protein, contributed both maternally and zygotically, is necessary and sufficient to specify and maintain the C blastomere lineage in the C. elegans embryo (Hunter and Kenyon, 1996). A number of this master regulator's targets were identified by microarrays comparing the transcript abundance in wild-type and mutant embryos either lacking or containing extra C blastomeres. Furthermore, we collected these embryos at defined time points, thus additionally providing temporal information. Target genes could then be separated by their transcriptional initiation into four consecutive temporal phases defined by a singular cell cycle beginning with the 2C-cell stage (Baugh et al, 2003). Using reporter YFP constructs for thirteen of the targets and a volume-rendering program, the 3D spatial expression pattern of each target gene was established. On the basis of this spatial information and knowledge of the temporal phase to which each target belongs, we have proposed a set of regulatory relationships between the components. We are currently testing these hypotheses by disrupting potential (capital O, grave accent)upstream(capital O, acute accent) regulators via RNAi and/or mutation and either observing the effect on individual (capital O, grave accent)downstream(capital O, acute accent) reporters or analyzing the effect on transcript abundance using QPCR. We hope that such measurements will give us insight into how the genes within the pal-1 network regulate each other in order to establish and maintain the various cell fates within the C blastomere lineage. Hunter, C.P. and Kenyon, C. (1996). Spatial and temporal controls target pal-1 blastomere-specification activity to a single blastomere lineage in C. elegans embryos. Cell 87, 217-26. Baugh, L.R., Hill, A.A., Slonim, D.K., Brown, E.L. and Hunter, C.P. (2003). Composition and dynamics of the Caenorhabditis elegans early embryonic transcriptome. Development 130, 889-900.

Tawe WN et al. (1997) Worm Breeder's Gazette "Paraquat mediates differential gene expression in C. elegans"

Henkle-Duehrsen K, Tawe WN, Eschbach M-L, Walter RD

[Worm Breeder's Gazette, 1997]

In order to identify genes that are differentially expressed as a consequence of stress due to paraquat, we used the differential display technique (1) to compare mRNA expression patterns in Caenorhabditis elegans. A C. elegans mixed-stage worm population, as well as a homogeneous larval population were treated with 100 mM paraquat, parallel to controls. Over 50 mRNA species that are potentially up-regulated in response to paraquat were identified. Sixteen of these candidates were re-amplified, ligated into plasmid vectors and the nucleotide sequences were determined. The induction of four of these expressed sequence tags (ESTs) designated L1, M47, M96 and M132 were confirmed in two independent stress / differential display experiments, as well as by northern blot analysis with RNA from stressed and unstressed worms. The nucleotide sequences of the independently isolated L1 and M47 ESTs were found to be identical. Their corresponding mRNA level increased more than 40-fold in the larval stage, and to a lesser extent in the mixed-stage worm population, in response to paraquat. Induction levels of 3 - 5 fold were observed for the M132 and M96 ESTs, in both larval and mixed populations. All of the isolated ESTs showed homology to portions of the C. elegans cosmids. The L1/M47 EST is derived from a gene encoding one of the putative C. elegans glutathione S-transferases. The paraquat-inducible M132 EST was also identified, and encodes a putative C2H2 - type zinc finger protein, possessing an N-terminal leucine zipper. However, the M96 EST appears to be a novel gene whose product has not yet been identified. Searches of other eukaryotic nucleotide sequence databases did not reveal any significant homologies to known sequences from any organism. Since paraquat is known to generate superoxide radicals in vivo, and the cellular superoxide dismutase (SOD) enzyme complex is responsible for the quenching of the deleterious effects of these radicals, the response of the C. elegans superoxide dismutases (2,3,4) to paraquat was also investigated in this study. Northern blot experiments demonstrated that mRNA steady state levels of the C. elegans manganese type and the copper/zinc type superoxide dismutases increased two-fold in response to paraquat, in the larval population. In contrast, mixed-stage populations did not show any apparent increase in the levels of these SOD mRNAs in response to paraquat. References: 1. Liang, P., Pardee, A.B. (1992). Science 257: 967 - 971 2. Giglio, M-P., Hunter, T., Bannister, J.V., Bannister, W.H., Hunter, G.J. (1994). Biochem. Mol. Biol. Int. 33 (1): 37-40 3. Giglio, A. M., Hunter, T., Bannister, J.V., Bannister, W.H., Hunter, G.J. (1994) . Biochem. Mol. Biol. Int. 33 (1): 37-40 4. Larsen, P.L. (1993). Proc. Natl. Acad. Sci. USA, 90: 8905-8909

Joanne C Wen et al. (2003) International Worm Meeting "Functional Characterization of Early Embryonic PAL-1 Targets"

Craig P Hunter, Joanne C Wen, L Ryan Baugh

[International Worm Meeting, 2003]

The activation and maintenance of C lineage specification occurs through maternal and zygotic PAL-1 activity, respectively (Hunter & Kenyon, 1996; Edgar et al, 2001). A set of targets of this master regulatory transcription factor were identified by transcript profiling embryos with perturbed PAL-1 activity (see abstract by Baugh et al). To functionally characterize PAL-1 targets, we have used RNAi to assess the lethality and terminal phenotypes following loss of function. To identify interactions between targets, we are performing epistasis analysis both by scoring synthetic lethality and by examining the effect of RNAi against one target on the expression of reporters for other targets. Our hope is that such functional characterization of a key set of PAL-1 targets will generate the data necessary to begin modeling the PAL-1 regulatory network.

Johnson TE (2001) Genome Res "SAGE thoughts on aging."

Johnson TE

[Genome Res, 2001]

The scientific method, and genetic analysis in particular, is based upon identifying variations between individuals of the same species. The study of Jones et al. in this issue reveals variation in transcript abundance between two developmental stages of the nematode Caenorhabditis elegns. In this case, the variation is not genetically specified ut is induced bt the environment as part of a shift to an alternate developmental form, the daur larva. In this type of whole-genome analyses, it is assumed that such studies would reveal differences in transcript abundance that would be casusall associated with distinct molecular and morphological transformations driving development. Much of this paper is conjecture about how the observed differences in transcriipt abundance specify observed differences in longevity(or, more precisely, in mortality rate).

Sorensen, Erika et al. (2019) International Worm Meeting "MicroRNA depletion results in Notch loss of function phenotypes and loss of stem cell totipotency."

Ballard, Joseph, Kimble, Judith, Jones, Hunter, Sorensen, Erika, Sorokin, Elena, Geier, Ben Geier, Groth, Amy Groth, Doenier, Emma

[International Worm Meeting, 2019]

Notch signaling regulates stem cells and differentiation during normal animal development and when dysregulated can lead to cancer. In the model organism C. elegans, Notch signaling functions to maintain germline stem cells (GSCs) in a totipotent state (capable of differentiating into all cell types) by promoting the expression of target genes that function in GSC maintenance. Recently, microRNAs (miRNAs) mir-61 and mir-250, termed collectively mir61-250, were identified as Notch target genes in GSCs. miRNAs are non-coding RNAs that act post-transcriptionally to limit the expression of other genes. To ask if mir61-250 affects GSC maintenance, we performed assays using a CRISPR-Cas9 generated mutant, which lacks the mir61-250 promoter. Unfortunately, no GSC defect was observed. This lack of defect is consistent with previous reports which show that single-gene miRNA deletions fail to produce strong mutant phenotypes unless placed in a sensitized background. Progressing forward, our lab has placed mir61-250 mutant animals in sensitized backgrounds and assayed them for defects in animal development, GSCs, and fertility. We find that mir61-250 mutants exhibit developmental deformities in their egg laying apparatus and have fewer GSCs. In addition, we find that loss of these miRNAs promotes reprograming from a germ cell fate to a somatic fate.

James F Cregg et al. (2004) West Coast Worm Meeting "A RNAi Screen for Genes Involved in Tolerance to Nicotine"

Kathleen B Quast, Kevin J Ford, William R Schafer, James F Cregg, Caroline R Craig, Suchitra Jagannathan

[West Coast Worm Meeting, 2004]

Tobacco use is the second major cause of death worldwide. The acquisition of tolerance to nicotine is a key step in the development of nicotine addiction. Twenty seven nicotinic acetylcholine receptor subunits have been identified in C. elegans (reviewed in Jones and Sattelle, 2003). However, few of the molecules which modify nAChR function, abundance, or subcellular localization in response to nicotine exposure are known. We are performing a RNAi-based screen of the first chromosome using the Ahringer lab RNAi feeding library in a rrf-3 background. In preliminary experiments, worms fed RNAi against genes already known to be involved in nicotine responces (including unc-63 and unc-50) have shown resistance in our nicotine-induced paralysis assay. Results of this screen should provide insight into the mechanisms of nicotine tolerance.