Localized translation in mammalian dendrites may play a role in synaptic plasticity and contribute to the molecular basis for learning and memory. The regulatory mechanisms that control localized translation in neurons are not well understood. We propose a role for microRNAs (miRNAs), a class of noncoding RNAs, as mediators of neuronal translational regulation. We have identified 86 miRNAs expressed in mammalian neurons, of which 40 have not previously been reported. A subset of these miRNAs exhibits temporally regulated expression in cortical cultures. Moreover, all of the miRNAs that were tested cofractionate with polyribosomes, the sites of active translation. These findings indicate that a large, diverse population of miRNAs may function to regulate translation in mammalian neurons.translational control ͉ noncoding RNA ͉ messenger ribonucleoprotein complex (mRNP)

MicroRNAs (miRNAs) constitute an extensive class of noncoding RNAs that are thought to regulate the expression of target genes via complementary base-pair interactions. To date, cloning has identified over 200 miRNAs from diverse eukaryotic organisms. Despite their success, such biochemical approaches are skewed toward identifying abundant miRNAs, unlike genome-wide, sequence-based computational predictions. We developed informatic methods to predict miRNAs in the C. elegans genome using sequence conservation and structural similarity to known miRNAs and generated 214 candidates. We confirmed the expression of four new miRNAs by Northern blotting and used a more sensitive PCR approach to verify the expression of ten additional candidates. Based on hypotheses underlying our computational methods, we estimate that the C. elegans genome may encode between 140 and 300 miRNAs and potentially many more.

Genetic and biochemical analyses of RNA interference (RNAi) and microRNA (miRNA) pathways have revealed proteins such as Argonaute/PIWI and Dicer that process and present small RNAs to their targets. Well validated small RNA pathway cofactors, such as the Argonaute/PIWI proteins show distinctive patterns of conservation or divergence in particular animal, plant, fungal, and protist species. We compared 86 divergent eukaryotic genome sequences to discern sets of proteins that show similar phylogenetic profiles with known small RNA cofactors. A large set of additional candidate small RNA cofactors have emerged from functional genomic screens for defects in miRNA- or siRNA-mediated repression in C. elegans and D. melanogaster1,2 and from proteomic analyses of proteins co-purifying with validated small RNA pathway proteins3,4. The phylogenetic profiles of many of these candidate small RNA pathway proteins are similar to those of known small RNA cofactor proteins. We used a Bayesian approach to integrate the phylogenetic profile analysis with predictions from diverse transcriptional coregulation and proteome interaction datasets to assign a probability for each protein for a role in a small RNA pathway. Testing high-confidence candidates from this analysis for defects in RNAi silencing, we found that about half of the predicted small RNA cofactors are required for RNAi silencing. Many of the newly identified small RNA pathway proteins are orthologues of proteins implicated in RNA splicing. In support of a deep connection between the mechanism of RNA splicing and small RNA-mediated gene silencing, the presence of the Argonaute proteins and other small RNA components in the many species analysed strongly correlates with the number of introns in that species.

The let-7 microRNA (miRNA) gene of Caenorhabditis elegans controls the timing of developmental events. let-7 is conserved throughout bilaterian phylogeny and has multiple paralogs. Here, we show that the paralog mir-84 acts synergistically with let-7 to promote terminal differentiation of the hypodermis and the cessation of molting in C. elegans. Loss of mir-84 exacerbates phenotypes caused by mutations in let-7, whereas increased expression of mir-84 suppresses a let-7 null allele. Adults with reduced levels of mir-84 and let-7 express genes characteristic of larval molting as they initiate a supernumerary molt. mir-84 and let-7 promote exit from the molting cycle by regulating targets in the heterochronic pathway and also nhr-23 and nhr-25, genes encoding conserved nuclear hormone receptors essential for larval molting. The synergistic action of miRNA paralogs in development may be a general feature of the diversified miRNA gene family.

The Caenorhabditis elegans microRNAs (miRNAs) lin-4 and let-7 promote transitions between stage-specific events in development by down-regulating the translation of their target genes. Expression of let-7 is required at the fourth larval stage for the proper transition from larval to differentiated, adult fates in the hypodermis; however, it was not known whether expression of let-7 is sufficient to specify these adult fates. To test this, we created fusion genes between lin-4 and let-7 that direct the expression of let-7 two stages early, at the L2 stage. We find that animals bearing the fusion genes show precocious adult development at the L4 stage, indicating that temporal misexpression of let-7 is sufficient to direct the larval-to-adult transition. Additionally, an RNA interference (RNAi)-based screen for enhancers of the precocious phenotype identified the period ortholog lin-42, among other genes, which are candidate modulators of the effects of let-7 expression. let-7 is conserved throughout bilaterian phylogeny, and orthologs of its targets have roles in vertebrate development, suggesting the importance of understanding how let-7 promotes terminal differentiation in C. elegans and other organisms.

Hundreds of microRNAs (miRNAs) have been discovered in metazoans and plants, and understanding of their biogenesis has advanced dramatically; however, relatively little is known about the cofactors necessary for miRNA regulation of target gene expression. In Caenorhabditis elegans, the conserved miRNA let-7 and its paralogs, including mir-84, control the timing of stage-specific developmental events. To identify factors required for the activity of mir-84 and possibly other miRNAs, we screened for mutations that suppress the developmental defects caused by overexpression of mir-84. Mutations in the somi-1 gene prevent these defects without affecting the expression level of mir-84. Loss of somi-1 also causes phenotypes similar to deletion of mir-84, showing that somi-1 is necessary for the normal function of this miRNA. somi-1 encodes a zinc finger protein that localizes to nuclear foci and binds the promoters of let-60/RAS, lin-14, and lin-28, genes that may be targeted by mir-84 and similar miRNAs. Genetic evidence shows that somi-1 inhibits lin-14 and induction of the vulval precursors by the let-60/RAS pathway. Proteomic and genetic screens identified conserved chromatin-remodeling and homeodomain transcription factor complexes that work with somi-1 to regulate differentiation. Our results suggest that somi-1 coordinates a nuclear response that complements the activity of mir-84.

microRNAs (miRNAs) are ∼22 nt regulatory RNAs that in animals typically bind with partial complementarity to sequences in the 3′ untranslated (UTR) regions of target mRNAs, to induce a decrease in the production of the encoded protein. The relative contributions of translational inhibition of intact mRNAs and degradation of mRNAs caused by binding of the miRNA vary; for many genetically validated miRNA targets, translational repression has been implicated, whereas some analyses of other miRNA targets have revealed only modest translational repression and more significant mRNA destabilization. In Caenorhabditis elegans, the lin-4 miRNA accumulates during early larval development, binds to target elements in the lin-14 mRNA, and causes a sharp decrease in the abundance of LIN-14 protein. Here, we monitor the dynamics of lin-14 mRNA and protein as well as lin-4 miRNA levels in finely staged animals during early larval development. We find complex regulation of lin-14, with the abundance of lin-14 mRNA initially modestly declining followed by fluctuation but little further decline of lin-14 mRNA levels accompanied by continuing and more dramatic decline in LIN-14 protein abundance. We show that the translational inhibition of lin-14 is dependent on binding of the lin-4 miRNA to multiple lin-4 complementary sites in the lin-14 3′UTR. Our results point to the importance of translational inhibition in silencing of lin-14 by the lin-4 miRNA.