RESUMO
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.
Assuntos
Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , RNA de Helmintos/genética , RNA de Helmintos/metabolismo , Envelhecimento/genética , Envelhecimento/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/crescimento & desenvolvimento , Proteínas de Caenorhabditis elegans/química , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genes de Helmintos , Insulina/metabolismo , Fator de Crescimento Insulin-Like I/metabolismo , Longevidade/genética , Longevidade/fisiologia , Dados de Sequência Molecular , Mutação , Estrutura Terciária de Proteína , Estabilidade de RNA , Receptor de Insulina/genética , Receptor de Insulina/metabolismo , Homologia de Sequência de Aminoácidos , Transdução de Sinais , TemperaturaRESUMO
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.
Assuntos
Genes de Helmintos , Planárias/genética , Planárias/fisiologia , Regeneração/genética , Regeneração/fisiologia , Animais , Padronização Corporal/genética , Padronização Corporal/fisiologia , Diferenciação Celular , Sequência Conservada , DNA de Helmintos/genética , Testes Genéticos , Homeostase , Fenótipo , Planárias/citologia , Planárias/crescimento & desenvolvimento , Interferência de RNA , Células-Tronco/fisiologia , Células-Tronco/efeitos da radiação , Terminologia como Assunto , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
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.
Assuntos
Antinematódeos/farmacologia , Betaína/metabolismo , Betaína/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Sistema Nervoso/metabolismo , Animais , Animais Geneticamente Modificados , Antinematódeos/metabolismo , Tamanho Corporal/genética , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Relação Dose-Resposta a Droga , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Ativação do Canal Iônico/genética , Larva , Canais Iônicos de Abertura Ativada por Ligante/genética , Mecanorreceptores/efeitos dos fármacos , Mecanorreceptores/metabolismo , Potenciais da Membrana/efeitos dos fármacos , Mutação/genética , Neurotransmissores/farmacologia , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismoRESUMO
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.