Gary Ruvkun - US grants

Molecular genetic studies in Caenorhabditis elegans have revealed that heterochronic genes explicitly define and interpret temporal information during development. These genes control the temporal sequence of the C. elegans postembryonic cell lineage by generating a temporal molecular gradient of the two nuclear proteins that are the products of the heterochronic gene lin-14. We propose to dissect how the lin-14 gene generates and interprets temporal signals to control C. elegans development. We will establish how transcriptional and posttranscriptional regulation of lin-14 gene activity generates the temporal gradient reverse genetic analysis of mutations in lin-14 regulatory regions. By monitoring the regulation of the lin-14 gene in a variety of heterochronic mutants, we will dissect how these genes conspire with lin-14 to generate or interpret temporal information. We will explore the lin-14 biochemical function in the nucleus by testing whether lin-14 proteins can bind to the DNA or RNA of the other cloned heterochronic genes that genetic and molecular epistasis analysis suggests they may regulate. These interactions will be analyzed with various lin-14 protein mutants constructed in vitro. We will test whether each of the two lin-14 proteins specify distinct cell fates by expressing each inappropriately and scoring their effect on the fates of cells that express them. We will characterize a newly identified set of heterochronic genes that control timing of the neuron-regulated dauer developmental pathway. These heterochronic genes may control timing in the specific neuronal cells that control entry into the dauer stage, or they may be more general developmental timing genes. We will explore how these new heterochronic genes fit into the heterochronic gene epistasis pathway and into the dauer epistasis pathway to discern which genes they regulate or vice versa. We will generate transposon insertion mutations in these genes to facilitate their cloning and molecular analysis. The heterochrony observed in phylogeny is very reminiscent of that we have dissected in C. elegans and suggests that heterochronic genes like lin-14 may be major players in evolutionary change and developmental control across phylogeny. Based on numerous examples of conservation of gene structure and function between species, molecular and genetic explorations of these genes in C. elegans promises to illuminate the functions and mechanisms of their human homologues. Mutations in these human temporal control genes may be the molecular basis for some human genetic diseases (for example, particular cancers) that lead to temporal transformations in cell fate.

DESCRIPTION (Adapted from Applicant's Abstract): Molecular genetic studies in C. elegans have revealed that heterochronic genes explicitly control the temporal sequence of cell lineage by generating a temporal molecular gradient of the nuclear protein products of the heterochronic gene lin-14. Conserved elements in the lin-14 3' UTR that are complementary to the RNA products of the regulatory gene lin-4 generate this temporal gradient. By analysis of mutations that perturb the structure and placement of the multiple lin-4/lin- 14 RNA duplexes and other conserved elements, the applicant proposes to dissect how the lin-14 3' UTR generates the temporal gradient. He will assay the mutant lin-4/lin-14 RNA duplexes in an in vitro translation system which partially recapitulates lin-4 in vivo regulation of lin-14 3' UTR. He will correlate the in vivo and in vitro effects. He will use genetic screens to identify gene products that recognize the lin-4/lin-14 RNA duplex as well as other conserved elements to mediate temporal gradient formation. He will clone those genes that act in concert with lin-4 to down-regulate lin-14. He will determine the function of each LIN-14 protein by a combination of observation and ectopic expression experiments. He will use other genetic screens that will identify genes that act in combination with lin-14 or downstream of lin-14 to control the temporal fates of cells. Genes that act downstream of lin-14 will be cloned and the molecular mechanisms by which they are regulated by lin-14 will be studied. He will screen for proteins that interact with LIN-14 and characterize their function in temporal pattern formation by reverse genetic analysis. Regulation of pattern formation gene expression by 3' UTR regulation has been found in a number of systems, especially those involving the temporal and spatial cascade of maternal mRNA translational control. The involvement of an antisense RNA in this process is at this point unique in metazoans but likely to be the first example of a more generally used mechanism. In addition, the mechanisms and indeed the genetic components of the lin-14 temporal gradient pathway promise to allow the identification of homologues from other organisms such as man. The applicant posits that such homologues will perform very similar functions in temporal pattern formation or translational control. The recent discoveries of universal components in developmental control by, for example, HOX clusters and signalling pathways endorse such a view of universality. If the gene pathway is so conserved, mutations in human heterochronic genes may be molecular bases for various oncogenic and endocrine genetic diseases that transform cells to precursor or descendent fates.

The nematode C. elegans secretes a pheromone that controls whether the animal molts into the developmentally arrested, extremely long-lived dauer diapause stage, or into a non-arrested normally aging animal. The dauer pheromone signal is detected by sensory neurons which couple by an unknown neurosecretory mechanism to a response pathway in the various target tissues that are remodeled during the dauer stage. The neuronally- regulated developmental and neuroendocrine changes associated with dauer formation are a model for the hypothalmic/pituitary/somatic and autonomic nervous system signaling axes in vertebrates. We have genetically identified a number of genes that regulate the development or function of the dauer sensory to secretory neural pathway. We propose a molecular analysis of three of these genes, daf2, daf-16, and daf-23. Mutations in daf-2 and daf-23 also cause two to three fold increases in longevity of non-dauer animals, and mutations in daf-16 suppress this increase in longevity. Thus daf-2, daf-16 and daf23 not only regulate diapause, they also regulate senescence. We have shown that daf-23 encodes a phosphatidyl inositol-3 kinase, PI-3 kinase, a protein known from vertebrates to associate with receptor and non-receptor tyrosine kinases. We have shown that the mammalian homologue of DAF-23 can fully complement a daf-23 mutant. PI-3 kinase generates a membrane-bound signaling molecule, phosphatidyl inositol-3,4,5-P3, which couples to unknown effector molecules. Thus daf-23 could control dauer formation and senescence by coupling a tyrosine kinase to downstream effectors, either during development of the nervous system or during pheromone signaling. Our genetic analysis suggests that daf-2 and daf-16 encode proteins that transduce the phosphatidyl inositol signal generated by daf-23. We propose to study how the genes in &his pathway couple phosphatidyl inositol biosynthesis to control neuroendocrine function during diapause. Our analysis of the molecular basis of neurosecretory function in C. elegans could reveal the genes that control the development and function of human neurosecretory pathways, with implication for therapies in diabetes, metabolic disorders, and high blood pressure. In addition, since diapause represents a neurosecretory function that is used across invertebrate phylogeny, including in insect pests, new insecticides designed to inhibit P1P3 signaling and thus induce diapause, with its concomitant suspension of feeding and reproduction, would have important implications for agriculture and indirectly, human health and welfare. Our molecular analysis of senescence in C. elegans may also be broadly applicable to human aging. The long lifespan that is caused by a decrease in PI-3 kinase activity suggests that senescence in C. elegans is regulated by the production of phosphatidyl inositol-3,4,5-P3. Drugs which antagonize the production or activity of this lipid might increase longevity.

An insulin signaling pathway couples feeding and nutritional status in mammals to the rate and mode of metabolism in most tissues of the animal. We have shown that an insulin-like signaling pathway regulates longevity and metabolism in C. elegans. This is reminiscent and may be mechanistically related to the longevity increase caused by caloric restriction in mammals. Thus the genetic components of the C. elegans insulin signaling pathway may be key components of a mammalian longevity determining pathway. Our genetic analysis has also revealed that the key output of C. elegans insulin-like signaling is the activity of the transcription factor DAF-16. Much of this proposal focuses on how DAF-2 insulin-like receptor signals are transduced to the DAF-16 transcription factor, and how those modulations of DAF-16 activity in turn regulate metabolism and longevity. Three human homologues of DAF-16 are excellent candidates for transducing insulin-like signaling in humans to also regulate longevity and metabolism. We will test whether these human proteins can function in the C. elegans insulin-like signaling pathway, that is, are functional homologues. In addition to their possible roles in longevity control, the insulin signaling genes we have identified by C. elegans genetics may reveal components of insulin signaling in mammals that are important for the understanding and eventual treatment of diabetes. Diabetes is a common diseases that affects the production or response to insulin, causing devastating metabolic dysregulations. The molecular basis of the defective insulin response in the adult onset or type II diabetes is unknown. It is clear that it is at least in part a genetic disease. The C. elegans genetics strongly argues that inhibition of human DAF-16 activity by drugs may bypass the need for upstream insulin signaling. Thus human DAF-16 may become a major target for pharmaceutical development of diabetes therapies.

Explicit genetic control of temporal patterning has been revealed by analysis of the heterochronic gene pathway in C. elegans. Depending on the mutation, stage-specific developmental events in heterochronic mutant animals occur at either earlier or later stages than they would normally. Two small regulatory RNAs control successive steps in this pathway by regulating the activities of key target mRNAs that encode cell fate determination factors. Both regulatory RNAs are complementary to these target heterochronic genes. In one case, this regulation has been shown to occur at a post-transcriptional step. We propose to discern the mechanism by which these regulatory RNAs control the translation of their mRNA targets. We will use a combination of genetic and biochemical techniques in this analysis. The discovery of two regulatory RNAs in the heterochronic pathway suggests the attractive model that the expression of a regulatory RNA at each larval molt may trigger the down-regulation of particular stage-specific regulatory proteins. Regulatory RNAs like lin-4 and let-7 may also be more general in animal species and may perform the same biochemical function in translational control. We have detected possible let-7 homologues in Drosophila and human genome databases, suggesting that its function may be more ancient than the common ancestor of molting animals. Noncoding RNAs in eukaryotic organisms are involved in translation (the ribosomal and tRNAs), RNA processing (the Un RNAs) and RNA methylation/pseudouridination (the snoRNAs). Regulatory RNAs such as let-7 tend to be missed by genome sequence analysis and this is a major problem for genomics. Our analysis of the key features of these regulatory RNAs, their promoter elements, the key loops and bulges for function, the regions complementary to target mRNAs, may enable the informatic detection of analogous but not homologous regulatory RNAs in genome databases.

An insulin signaling pathway couples feeding and nutritional status in mammals to metabolism in most tissues. An insulin-like signaling pathway regulates longevity and metabolism in C. elegans. This is reminiscent and may be mechanistically related to the longevity increase caused by caloric restriction in mammals. Our genetic analysis has also revealed that the signal transduction components and transcriptional outputs of C. elegans insulin-like signaling pathway. Mammalian orthologs of many of these genes have been identified. Thus the genetic components of the C. etegans insulin signaling pathway may be key components of a mammalian longevity determining pathway. While many points of congruence have been identified, we know from genetic epistasis experiments that there are missing components. We will identify these components by second generation genetics, using the mutants identified in the first round of genetics as tools in the genetics of this round. We will also explore the connection between reproductive longevity and organismal longevity by genetically analysing the regulation of reproductive senescence in C. elegans. We have shown that C. elegans has a large family of insulin like hormones and that human insulin will function in C. elegans. Our genetics has revealed that not all responses to C. elegans as well as human insulin are mediated by the canonical insulin response receptors. We will explore the novel insulin response pathway by genetic analysis. We will determine the molecular identity of the worm genes revealed by the extensive genetic analysis proposed in this grant, search for human homologues of those genes, and test whether these human proteins in fact can function in the C. elegans insulin-like signaling pathway, that is, are functional homologues. In addition to their possible roles in longevity control, the insulin signaling genes we have identified by C. elegans genetics may reveal components of insulin signaling in mammals that are important for the understanding and eventual treatment of diabetes. Diabetes is a common disease that affects the production or response to insulin, causing devastating metabolic dysregulations. The molecular basis of the defective insulin response in the adult onset or type IIdiabetes is unknown. It is clear that it is at least in part a genetic disease. Saturation genetic analysis of the homologous C. elegans metabolic control pathway has revealed genes that act downstream of the insulin-like receptor as well as other neuroendocrine signals that converge with insulin. The products of the genes we have identified may be targets for pharmaceutical development of diabetes therapies.

DESCRIPTION (provided by applicant): We have identified a number of genes via genetic analysis and RNA interference gene inactivations that act as protein coding cofactors for the function of miRNAs and siRNAs in C. elegans. Some of these proteins were identified in genetic screens for decrease in miRNA function, some in genetic screens for decrease in siRNA function, and some in genetic screens for increase in siRNA function. We have also identified the target small RNAs that mediate these functions by deep RNA sequencing of selected mutant strains. We propose to dissect in detail how these proteins orchestrate the production, trafficking, and function of small RNAs in both mRNA degradation, mRNA translational control, and control of gene expression. We also propose to discern how the miRNA and siRNA and other small RNA pathways may compete with each other for common cofactors, thus leading to an increase in function in one pathway, when the other pathway is debilitated. The genes identified in this study are likely to be key factors in the function of small RNAs in biology and thus their identification may enable more potent RNAi based drug development. PUBLIC HEALTH RELEVANCE: The Ruvkun lab has identified a large collection of genes that act as cofactors for the function of small RNA genes in C. elegans. We propose to dissect in detail how these proteins orchestrate the production, trafficking, and function of small RNAs in control of gene expression. The genes identified in this study are likely to be key factors in the function of small RNAs in biology and thus their identification may enable more potent RNA interference-based drug development.

DESCRIPTION (provided by applicant): From a combination of genetic analyses and RNAi screening, we have discovered hundreds of gene inactivations and mutations that promise to reveal the neuroendocrine circuit through which C. elegans fat storage set points are determined. Because RNAi does not as potently target neurons, we have also configured classical genetic screens for low fat storage and high fat storage mutations. Some of the 80 mutants in our current collection store extraordinarily high levels of fat under all conditions tested whereas others have defects in the mobilization of fat induced by starvation or drug treatments. Interestingly, many of the mutants have defects in the behavioral outputs normally induced by starvation or satiety. Thus these mutants do not feel starved. We propose to molecular identify 5 of these top candidate mutants per year to discern their molecular identity as well as to delineate from their expression pattern which cells mediate the assessment of fat and the behaviors that drive fat storage. We will determine which of these gene inactivations affect fat levels by regulating rates of feeding and which affect gross metabolic levels. We will determine the cellular focus of gene activity for fat storage and whether any of the genes encode proteins that mediate the actual sorting of fats in the storage organs. From a gene array of starved and well fed animals, we have also discovered a number of genes that are induced by starvation or by feeding. GFP fusions to these genes have generated a robust set of reporters of the starved state. We will cross these reporter genes into our mutant collection to assess which mutants induce a starved state and which do not. In addition, we have used these reporter genes already in a classical genetic screen for mutants that fail to induce a starvation marker gene. C. elegans is amenable to large scale genetic and functional genomic screens which is not feasible in mice. Our worm genomics highlights scores of human genes which are homologues of the worm genes we have identified. In some cases, the genes encode proteins that are attractive for the development of drugs. Therefore, identification of fat storage pathway genes in C. elegans provides targets for intervention of human obesity. PUBLIC HEALTH RELEVANCE Our C. elegans gene inactivation analysis has revealed hundreds of genes that regulate of fat storage, many of which have human homologues. The studies of obesity in C. elegans is likely to identify targets for intervention of obesity in human.

DESCRIPTION (provided by applicant): We have preliminary evidence that small RNAs of the size of siRNA and miRNAs are coupled to modified nucleotides such as coenzyme A and bis-MGD. These modifications would allow small RNAs to covalently engage with the proteome to constitute nucleic acid bar codes on these proteins. We propose to use powerful nucleic acid hybridization and selection techniques to purify the proteins covalently linked to RNA and to use mass spectroscopy to identify those linked proteins. Using RNAi of these proteins we will survey for defects in small RNA function and thus an involvement of these proteins in the functions of small RNAs. Because all of the modifications are predicted to occur at the 5' end of small RNAs, most of these modified RNAs will have been systematically missed by surveys most of which demands 5' OH or 5' phosphates on the RNAs for cloning.