Victor R. Ambros

The functions of animal microRNAs

MicroRNAs (miRNAs) are small RNAs that regulate the expression of complementary messenger RNAs. Hundreds of miRNA genes have been found in diverse animals, and many of these are phylogenetically conserved. With miRNA roles identified in developmental timing, cell death, cell proliferation, haematopoiesis and patterning of the nervous system, evidence is mounting that animal miRNAs are more numerous, and their regulatory impact more pervasive, than was previously suspected.

Efficient gene transfer in C.elegans: extrachromosomal maintenance and integration of transforming sequences.

We describe a dominant behavioral marker, rol‐6(su‐1006), and an efficient microinjection procedure which facilitate the recovery of Caenorhabditis elegans transformants. We use these tools to study the mechanism of C.elegans DNA transformation. By injecting mixtures of genetically marked DNA molecules, we show that large extrachromosomal arrays assemble directly from the injected molecules and that homologous recombination drives array assembly. Appropriately placed double‐strand breaks stimulated homologous recombination during array formation. Our data indicate that the size of the assembled transgenic structures determines whether or not they will be maintained extrachromosomally or lost. We show that low copy number extrachromosomal transformation can be achieved by adjusting the relative concentration of DNA molecules in the injection mixture. Integration of the injected DNA, though relatively rare, was reproducibly achieved when single‐stranded oligonucleotide was co‐injected with the double‐stranded DNA.

microRNAs: Tiny Regulators with Great Potential

Animal genomes contain an abundance of small genes that produce regulatory RNAs of about 22 nucleotides in length. These microRNAs are diverse in sequence and expression patterns, and are evolutionarily widespread, suggesting that they may participate in a wide range of genetic regulatory pathways.

MicroRNA Pathways in Flies and Worms: Growth, Death, Fat, Stress, and Timing

Drosophila geneticists have uncovered roles for microRNAs in the coordination of cell proliferation and cell death during development, and in stress resistance and fat metabolism. In C. elegans, a homolog of the well-known fly developmental regulator hunchback acts downstream of the microRNAs lin-4 and let-7 in a pathway controlling developmental timing.

Characterization of MicroRNA Expression Levels and Their Biological Correlates in Human Cancer Cell Lines

MicroRNAs are small noncoding RNAs that function by regulating target gene expression posttranscriptionally. They play a critical role in developmental and physiologic processes and are implicated in the pathogenesis of several human diseases including cancer. We examined the expression profiles of 241 human microRNAs in normal tissues and the NCI-60 panel of human tumor-derived cell lines. To quantify microRNA expression, we employed a highly sensitive technique that uses stem-loop primers for reverse transcription followed by real-time PCR. Most microRNAs were expressed at lower levels in tumor-derived cell lines compared with the corresponding normal tissue. Agglomerative hierarchical clustering analysis of microRNA expression revealed four groups among the NCI-60 cell lines consisting of hematologic, colon, central nervous system, and melanoma tumor-derived cell lines clustered in a manner that reflected their tissue of origin. We identified specific subsets of microRNAs that provide candidate molecular signatures characteristic of the tumor-derived cell lines belonging to these four clusters. We also identified specific microRNA expression patterns that correlated with the proliferation indices of the NCI-60 cell lines, and we developed evidence for the identification of specific microRNAs as candidate oncogenes and tumor suppressor genes in different tumor types. Our results provide evidence that microRNA expression patterns may mark specific biological characteristics of tumors and/or mediate biological activities important for the pathobiology of malignant tumors. These findings call attention to the potential of microRNAs to provide etiologic insights as well as to serve as both diagnostic markers and therapeutic targets for many different tumor types.

MicroRNAs and Other Tiny Endogenous RNAs in C. elegans

BACKGROUND MicroRNAs (miRNAs) are small noncoding RNAs that are processed from hairpin precursor transcripts by Dicer. miRNAs probably inhibit translation of mRNAs via imprecise antisense base-pairing. Small interfering RNAs (siRNAs) are similar in size to miRNAs, but they recognize targets by precise complementarity and elicit RNA-mediated interference (RNAi). We employed cDNA sequencing and comparative genomics to identify additional C. elegans small RNAs with properties similar to miRNAs and siRNAs. RESULTS We found three broad classes of small RNAs in C. elegans: (1) 21 new miRNA genes (we estimate that C. elegans contains approximately 100 distinct miRNA genes, about 30% of which are conserved in vertebrates; (2), 33 distinct members of a class of tiny noncoding RNA (tncRNA) genes with transcripts that are similar in length to miRNAs (approximately 20-21 nt) and that are in some cases developmentally regulated but are apparently not processed from a miRNA-like hairpin precursor and are not phylogenetically conserved; (3) more than 700 distinct small antisense RNAs, about 20 nt long, that are precisely complementary to protein coding regions of more than 500 different genes and therefore seem to be endogenous siRNAs. CONCLUSIONS The presence of diverse endogenous siRNAs in normal worms suggests ongoing, genome-wide gene silencing by RNAi. miRNAs and tncRNAs are not predicted to form complete Watson-Crick hybrids with any C. elegans RNA target, and so they are likely to regulate the activity of other genes by non-RNAi mechanisms. These results suggest that diverse modes of small RNA-mediated gene regulation are deployed in normal worms.

Most Caenorhabditis elegans microRNAs Are Individually Not Essential for Development or Viability

MicroRNAs (miRNAs), a large class of short noncoding RNAs found in many plants and animals, often act to post-transcriptionally inhibit gene expression. We report the generation of deletion mutations in 87 miRNA genes in Caenorhabditis elegans, expanding the number of mutated miRNA genes to 95, or 83% of known C. elegans miRNAs. We find that the majority of miRNAs are not essential for the viability or development of C. elegans, and mutations in most miRNA genes do not result in grossly abnormal phenotypes. These observations are consistent with the hypothesis that there is significant functional redundancy among miRNAs or among gene pathways regulated by miRNAs. This study represents the first comprehensive genetic analysis of miRNA function in any organism and provides a unique, permanent resource for the systematic study of miRNAs.

Potent effect of target structure on microRNA function

MicroRNAs (miRNAs) are small noncoding RNAs that repress protein synthesis by binding to target messenger RNAs. We investigated the effect of target secondary structure on the efficacy of repression by miRNAs. Using structures predicted by the Sfold program, we model the interaction between an miRNA and a target as a two-step hybridization reaction: nucleation at an accessible target site followed by hybrid elongation to disrupt local target secondary structure and form the complete miRNA-target duplex. This model accurately accounts for the sensitivity to repression by let-7 of various mutant forms of the Caenorhabditis elegans lin-41 3′ untranslated region and for other experimentally tested miRNA-target interactions in C. elegans and Drosophila melanogaster. These findings indicate a potent effect of target structure on target recognition by miRNAs and establish a structure-based framework for genome-wide identification of animal miRNA targets.

Temporal regulation of microRNA expression in Drosophila melanogaster mediated by hormonal signals and broad-Complex gene activity.

lin-4 and let-7 are founding members of an extensive family of genes that produce small transcripts, termed microRNAs (miRNAs). In Caenorhabditis elegans, lin-4 and let-7 control the timing of postembryonic events by translational repression of target genes, permitting progression from early to late developmental programs. To identify Drosophila melanogaster miRNAs that could play similar roles in the control of developmental timing, we characterized the developmental expression profile of 24 miRNAs in Drosophila, and found 7 miRNAs that are either upregulated or downregulated in conjunction with metamorphosis. The upregulation of three of these miRNAs (mir-100, mir-125, and let-7), and the downregulation of a fourth (mir-34) requires the hormone ecdysone (Ecd) and the activity of the Ecd-inducible gene Broad-Complex. Interestingly, mir-125 is a putative homologue of lin-4. mir-100, -125, and let-7 are clustered within an 800-bp region on chromosome 2L, suggesting that these three miRNAs may be coordinately regulated via common cis-acting elements during metamorphosis. In S2 cells, Ecd and the juvenile hormone analog methoprene exert opposite effects on the expression of these four miRNAs, indicating the participation of both these hormones in the temporal regulation of mir-34, -100, -125, and let-7 expression in vivo.

mirWIP: microRNA target prediction based on microRNA-containing ribonucleoprotein-enriched transcripts

Target prediction for animal microRNAs (miRNAs) has been hindered by the small number of verified targets available to evaluate the accuracy of predicted miRNA-target interactions. Recently, a dataset of 3,404 miRNA-associated mRNA transcripts was identified by immunoprecipitation of the RNA-induced silencing complex components AIN-1 and AIN-2. Our analysis of this AIN-IP dataset revealed enrichment for defining characteristics of functional miRNA-target interactions, including structural accessibility of target sequences, total free energy of miRNA-target hybridization and topology of base-pairing to the 5′ seed region of the miRNA. We used these enriched characteristics as the basis for a quantitative miRNA target prediction method, miRNA targets by weighting immunoprecipitation-enriched parameters (mirWIP), which optimizes sensitivity to verified miRNA-target interactions and specificity to the AIN-IP dataset. MirWIP can be used to capture all known conserved miRNA-mRNA target relationships in Caenorhabditis elegans at a lower false-positive rate than can the current standard methods.