Betaine acts on a ligand-gated ion channel in the nervous system of the nematode C. elegans.

Prior to the advent of synthetic nematocides, natural products such as seaweed were used to control nematode infestations. The nematocidal agent in seaweed is betaine, an amino acid that functions as an osmolyte and methyl donor. However, the molecular mechanisms of betaine toxicity are unknown. We identified the betaine transporter SNF-3 and the betaine receptor ACR-23 in the nematode C. elegans. Mutating snf-3 in a sensitized background caused the worms to be hypercontracted and paralyzed, presumably as a result of excess extracellular betaine. These behavioral defects were suppressed by mutations in acr-23, which encodes a ligand-gated cation channel of the cys-loop family. ACR-23 was activated by betaine and functioned in the mechanosensory neurons to maintain basal levels of locomotion. However, overactivation of the receptor by excess betaine or by the allosteric modulator monepantel resulted in hypercontraction and death of the nematode. Thus, monepantel targets a betaine signaling pathway in nematodes.

Post-developmental microRNA expression is required for normal physiology, and regulates aging in parallel to insulin/IGF-1 signaling in C. elegans.

Regulation of gene expression by microRNAs (miRNAs) is essential for normal development, but the roles of miRNAs in the physiology of adult animals are poorly understood. We have isolated a conditional allele of DGCR8/pash-1, which allows reversible and rapid inactivation of miRNA synthesis in vivo in Caenorhabditis elegans. This is a powerful new tool that allows dissection of post-developmental miRNA functions. We demonstrate that continuous synthesis of miRNAs is dispensable for cellular viability but critical for the physiology of adult animals. Loss of miRNA synthesis in the adult reduces lifespan and results in rapid aging. The insulin/IGF-1 signaling pathway is a critical determinant of lifespan, and is modulated by miRNAs. We find that although miRNA expression is required for some mechanisms of lifespan extension, it is not essential for the longevity of animals lacking insulin/IGF-1 signaling. Further, misregulated insulin/IGF-1 signaling cannot account for the reduced lifespan caused by disruption of miRNA synthesis. We show that miRNAs act in parallel with insulin/IGF-1 signaling to regulate a shared set of downstream genes important for physiological processes that determine lifespan. We conclude that coordinated transcriptional and post-transcriptional regulation of gene expression promotes longevity.

LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 microRNA processing in Caenorhabditis elegans.

The let-7 microRNA (miRNA) is an ultraconserved regulator of stem cell differentiation and developmental timing and a candidate tumor suppressor. Here we show that LIN-28 and the poly(U) polymerase PUP-2 regulate let-7 processing in Caenorhabditis elegans. We demonstrate that lin-28 is necessary and sufficient to block let-7 activity in vivo; LIN-28 directly binds let-7 pre-miRNA to prevent Dicer processing. Moreover, we have identified a poly(U) polymerase, PUP-2, which regulates the stability of LIN-28-blockaded let-7 pre-miRNA and contributes to LIN-28-dependent regulation of let-7 during development. We show that PUP-2 and LIN-28 interact directly, and that LIN-28 stimulates uridylation of let-7 pre-miRNA by PUP-2 in vitro. Our results demonstrate that LIN-28 and let-7 form an ancient regulatory switch, conserved from nematodes to humans, and provide insight into the mechanism of LIN-28 action in vivo. Uridylation by a PUP-2 ortholog might regulate let-7 and additional miRNAs in other species. Given the roles of Lin28 and let-7 in stem cell and cancer biology, we propose that such poly(U) polymerases are potential therapeutic targets.

Identification of genes needed for regeneration, stem cell function, and tissue homeostasis by systematic gene perturbation in planaria.

Planarians have been a classic model system for the study of regeneration, tissue homeostasis, and stem cell biology for over a century, but they have not historically been accessible to extensive genetic manipulation. Here we utilize RNA-mediated genetic interference (RNAi) to introduce large-scale gene inhibition studies to the classic planarian system. 1065 genes were screened. Phenotypes associated with the RNAi of 240 genes identify many specific defects in the process of regeneration and define the major categories of defects planarians display following gene perturbations. We assessed the effects of inhibiting genes with RNAi on tissue homeostasis in intact animals and stem cell (neoblast) proliferation in amputated animals identifying candidate stem cell, regeneration, and homeostasis regulators. Our study demonstrates the great potential of RNAi for the systematic exploration of gene function in understudied organisms and establishes planarians as a powerful model for the molecular genetic study of stem cells, regeneration, and tissue homeostasis.