Pheromones and Nutritional Signals Regulate the Developmental Reliance on let-7 Family MicroRNAs in C. elegans

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Solution structure of a let-7 miRNA:lin-41 mRNA complex from C. elegans

let-7 microRNA (miRNA) regulates heterochronic genes in developmental timing of the nematode Caenorhabditis elegans. Binding of miRNA to messenger RNA (mRNA) and structural features of the complex are crucial for gene silencing. We herein present the NMR solution structure of a model mimicking the interaction of let-7 miRNA with its complementary site (LCS 2) in the 3′ untranslated region (3′-UTR) of the lin-41 mRNA. A structural study was performed by NMR spectroscopy using NOE restraints, torsion angle restraints and residual dipolar couplings. The 33-nt RNA construct folds into a stem–loop structure that features two stem regions which are separated by an asymmetric internal loop. One of the stems comprises a GU wobble base pair, which does not alter its overall A-form RNA conformation. The asymmetric internal loop adopts a single, well-defined structure in which three uracils form a base triple, while two adenines form a base pair. The 3D structure of the construct gives insight into the structural aspects of interactions between let-7 miRNA and lin-41 mRNA.     

acn-1, a C. elegans homologue of ACE,genetically interacts with the let-7 microRNA and other heterochronic genes

The heterochronic pathway in C. elegans controls the relative timing of cell fate decisions during post-embryonic development. It includes a network of microRNAs (miRNAs), such as let-7, and protein-coding genes, such as the stemness factors, LIN-28 and LIN-41. Here we identified the acn-1 gene, a homologue of mammalian angiotensin-converting enzyme (ACE), as a new suppressor of the stem cell developmental defects of let-7 mutants. Since acn-1 null mutants die during early larval development, we used RNAi to characterize the role of acn-1 in C. elegans seam cell development, and determined its interaction with heterochronic factors, including let-7 and its downstream interactors – lin-41, hbl-1, and apl-1. We demonstrate that although RNAi knockdown of acn-1 is insufficient to cause heterochronic defects on its own, loss of acn-1 suppresses the retarded phenotypes of let-7 mutants and enhances the precocious phenotypes of hbl-1, though not lin-41, mutants. Conversely, the pattern of acn-1 expression, which oscillates during larval development, is disrupted by lin-41 mutants but not by hbl-1 mutants. Finally, we show that acn-1(RNAi) enhances the let-7-suppressing phenotypes caused by loss of apl-1, a homologue of the Alzheimer's disease-causing amyloid precursor protein (APP), while significantly disrupting the expression of apl-1 during the L4 larval stage. In conclusion, acn-1 interacts with heterochronic genes and appears to function downstream of let-7 and its target genes, including lin-41 and apl-1.     

The let-7/LIN-41 Pathway Regulates Reprogramming to Human Induced Pluripotent Stem Cells by Controlling Expression of Prodifferentiation Genes

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Expression and Preliminary Functional Profiling of the let-7 Family during Porcine Ovary Follicle Atresia

Most follicles in the mammalian ovary undergo atresia. Granulosa cell apoptosis is a hallmark of follicle atresia. Our previous study using a microRNA (miRNA) microarray showed that the let-7 microRNA family was differentially expressed during follicular atresia. However, whether the let-7 miRNA family members are related to porcine (Sus scrofa) ovary follicular apoptosis is unclear. In the current study, real-time quantitative polymerase chain reaction showed that the expression levels of let-7 family members in follicles and granulosa cells were similar to our microarray data, in which miRNAs let-7a, let-7b, let-7c, and let-7i were significantly decreased in early atretic and progressively atretic porcine ovary follicles compared with healthy follicles, while let-7g was highly expressed during follicle atresia. Furthermore, flow cytometric analysis and Hoechst33342 staining demonstrated that let-7g increased the apoptotic rate of cultured granulosa cells. In addition, let-7 target genes were predicted and annotated by TargetScan, PicTar, gene ontology and Kyoto encyclopedia of genes and genomes pathways. Our data provide new insight into the association between the let-7 miRNA family in granulosa cell programmed death.     

Developmental decisions: Balancing genetics and the environment by C. elegans

The small nematode C. elegans is characterized by developing through a highly coordinated, reproducible cell lineage that serves as the basis of many studies focusing on the development of multi-lineage organisms. Indeed, the reproducible cell lineage enables discovery of developmental defects that occur in even a single cell. Only recently has attention been focused on how these animals modify their genetically programmed cell lineages to adapt to altered environments. Here, we summarize the current understanding of how C. elegans responds to food deprivation by adapting their developmental program in order to conserve energy. In particular, we highlight the AMPK-mediated and insulin-like growth factor signaling pathways that are the principal regulators of induced cell cycle quiescence.     

Regulatory mutations of mir-48, a C. elegans let-7 family MicroRNA, cause developmental timing defects

The C. elegans heterochronic genes program stage-specific temporal identities in multiple tissues during larval development. These genes include the first two miRNA-encoding genes discovered, lin-4 and let-7. We show that lin-58 alleles, identified as lin-4 suppressors, define another miRNA that controls developmental time. These alleles are unique in that they contain point mutations in a gene regulatory element of mir-48, a let-7 family member. mir-48 is expressed prematurely in lin-58 mutants, whereas expression of mir-241, another let-7 family member residing immediately upstream of mir-48, appears to be unaffected. A mir-48 transgene bearing a lin-58 point mutation causes strong precocious phenotypes in the hypodermis and vulva when expressed from multicopy arrays. mir-48::gfp fusions reveal expression in these tissues, and inclusion of a lin-58 mutation causes precocious and enhanced gfp expression. These results suggest that lin-58 alleles disrupt a repressor binding site that restricts the time of miR-48 action in wild-type animals.