Publications

MicroRNAs (miRNAs) are regulatory RNAs found in multicellular eukaryotes, including humans, where they are implicated in cancer. The let-7 miRNA times seam cell terminal differentiation in C. elegans. Here we show that the let-7 family negatively regulates let-60/RAS. Loss of let-60/RAS suppresses let-7, and the let-60/RAS 3'UTR contains multiple let-7 complementary sites (LCSs), restricting reporter gene expression in a let-7-dependent manner. mir-84, a let-7 family member, is largely absent in vulval precursor cell P6.p at the time that let-60/RAS specifies the 1 degrees vulval fate in that cell, and mir-84 overexpression suppresses the multivulva phenotype of activating let-60/RAS mutations. The 3'UTRs of the human RAS genes contain multiple LCSs, allowing let-7 to regulate RAS expression. let-7 expression is lower in lung tumors than in normal lung tissue, while RAS protein is significantly higher in lung tumors, providing a possible mechanism for let-7 in cancer.

MicroRNAs (miRNAs) are small, non-coding regulatory RNAs found in many phyla that control such diverse events as development, metabolism, cell fate and cell death. They have also been implicated in human cancers. The C. elegans genome encodes hundreds of miRNAs, including the founding members of the miRNA family lin-4 and let-7. Despite the abundance of C. elegans miRNAs, few miRNA targets are known and little is known about the mechanism by which they function. However, C. elegans research continues to push the boundaries of discovery in this area. lin-4 and let-7 are the best understood miRNAs. They control the timing of adult cell fate determination in hypodermal cells by binding to partially complementary sites in the mRNA of key developmental regulators to repress protein expression. For example, lin-4 is predicted to bind to seven sites in the lin-14 3' untranslated region (UTR) to repress LIN-14, while let-7 is predicted to bind two let-7 complementary sites in the lin-41 3' UTR to down-regulate LIN-41. Two other miRNAs, lsy-6 and mir-273, control left-right asymmetry in neural development, and also target key developmental regulators for repression. Approximately one third of the C. elegans miRNAs are differentially expressed during development indicating a major role for miRNAs in C. elegans development. Given the remarkable conservation of developmental mechanism across phylogeny, many of the principles of miRNAs discovered in C. elegans are likely to be applicable to higher animals.

Caenorhabditis elegans let-7, a founding member of the microRNA family, is predicted to bind to six sites in the 3'UTR of the mRNA of its target gene, lin-41, to down-regulate LIN-41. Here, we demonstrate that wild-type let-7 microRNA binds in vitro to RNA from the lin-41 3'UTR. This interaction is dependent on two conserved let-7 complementary sites (LCSs). A 27-nucleotide sequence between the LCSs is also necessary for down-regulation in vivo. LCS mutations compensatory to the lesion in let-7(n2853) can partially restore lin-41 3'UTR function in a let-7(n2853) background, providing the first experimental evidence for an animal miRNA binding directly to its validated target in vivo.

MicroRNAs are small approximately 22 nucleotide regulators of numerous biological processes and bind target gene messenger RNAs to control gene expression. The C. elegans microRNA let-7 and its target lin-41 were the first microRNA::target interaction to be validated in vivo. let-7 molecules form imperfect duplexes with two required let-7 complementary sites in the lin-41 3' UTR. Here, we show that base pairing at both the 5' and 3' ends of the let-7 binding site, as well as the presence of unpaired RNA residues in the predicted duplexes, are required for lin-41 downregulation. In this study, our model for microRNA::target interactions also demonstrates that the context of a microRNA binding can be critical for function, revealing an unforeseen complexity in microRNA::target interactions.

MicroRNAs (miRNAs) are a large family of small regulatory RNAs that are poorly understood. The let-7 miRNA regulates the timing of the developmental switch from larval to adult cell fates during Caenorhabditis elegans development. Expression of let-7 RNA is temporally regulated, with robust expression in the fourth larval and adult stages. Here, we show that, like let-7 RNA, a transcriptional fusion of the let-7 promoter to gfp is temporally regulated, indicating that let-7 is transcriptionally controlled. Temporal upregulation of let-7 transcription requires an enhancer element, the temporal regulatory element (TRE), situated about 1200 base pairs upstream of the start of the mature let-7 RNA. The TRE is both necessary and sufficient for this temporal upregulation. A TRE binding factor (TREB) is able to bind to the TRE, and a 22-base pair inverted repeat within the TRE is necessary and sufficient for this binding. We also find that the nuclear hormone receptor DAF-12 and the RNA binding protein LIN-28 are both required for the correct timing of let-7 RNA and let-7::gfp expression. We speculate that these heterochronic genes regulate let-7 expression through its TRE.

hunchback regulates the temporal identity of neuroblasts in Drosophila. Here we show that hbl-1, the C. elegans hunchback ortholog, also controls temporal patterning. Furthermore, hbl-1 is a probable target of microRNA regulation through its 3'UTR. hbl-1 loss-of-function causes the precocious expression of adult seam cell fates. This phenotype is similar to loss-of-function of lin-41, a known target of the let-7 microRNA. Like lin-41 mutations, hbl-1 loss-of-function partially suppresses a let-7 mutation. The hbl-1 3'UTR is both necessary and sufficient to downregulate a reporter gene during development, and the let-7 and lin-4 microRNAs are both required for HBL-1/GFP downregulation. Multiple elements in the hbl-1 3'UTR show complementarity to regulatory microRNAs, suggesting that microRNAs directly control hbl-1. MicroRNAs may likewise function to regulate Drosophila hunchback during temporal patterning of the nervous system.

RNAi is routinely used to eliminate gene activity for experimental purposes. However, the precise molecular mechanism of RNAi is unknown. Recent papers partially illuminate this mechanism in human cells, advancing the potential application of RNAi toward the treatment of human disease.

Two small temporally regulated RNAs (stRNAs)* of approximately 22 nucleotides regulate timing of gene expression during development of the nematode C. elegans. This regulation occurs at a posttranscriptional, presumably translational, level and is distinct from RNA interference (RNAi). One of the two stRNAs, let-7, as well as its target gene, lin-41, are highly conserved even in humans, suggesting a wide employment of stRNA-mediated gene regulation. Recent reports indicate that these two stRNAs are indeed likely to represent only the tip of an iceberg with hundreds or more of additional micro-RNAs (miRNAs) existing in metazoans. miRNAs might thus be previously underestimated key participants in the field of gene regulation.