microRNAs
MicroRNAs (miRNA) are small RNAs of about 22 nucleotides in length. These tiny RNAs regulate the gene expression of target mRNAs by binding to the 3´ untranslated region (3´UTR), forming an RNA::RNA duplex. The mechanism of target gene repression is not well understood and recent studies demonstrate different consequences of the duplex formation for different miRNAs including translational repression and deadenylation of target mRNAs.
MicroRNAs were initially identified in C.elegans, but subsequently found in various animals, including humans, as well as plants. The biological functions of miRNAs in animals are diverse and include regulation of cell differentiation and proliferation, programmed cell death, organogenesis and metabolism. Numerous studies also implicate miRNAs in cancer, both as tumor suppressors and cancer-promoting "oncomiRs".
Biogenesis of miRNAs
The majority of miRNA genes are located in genomic regions distant from previously annotated genes and the biogenesis of these miRNAs has been studied in some detail (Fig.1). These miRNAs are transcribed from defined genetic loci by the RNA polymerase II as long, polyadenylated primary transcripts (Fig.1, step 1). In C.elegans, the primary transcripts of let-7 and possibly other miRNAs are trans-spliced, i.e., a short leader sequence named SL1 is added to the 5´end of the RNA (Fig.1, step 2). Trans-splicing occurs also during processing of the majority of mRNAs. The resulting pri-miRNAs are processed by the RNase III Drosha together with Pasha (partner of Drosha/DGCR8) into characteristic stem-loop structures of about 70 nt length, the so called pre-miRNAs (Fig.1, step 3). The stem-looped precursor is exported into the cytoplasm by Exportin-5 (Fig. 1, step 4). The pre-miRNA is subsequently processed by the RNase III Dicer to the mature miRNA of ~22 nt length (Fig.1, step 5). The binding to the 3´UTRs of mRNAs is imperfect and results in the repression of the target gene.
Figure 1 miRNA biogenesis
The biogenesis of miRNAs. MiRNAs are encoded by specific genetic loci and (1) transcribed by the RNA Polymerase II. The primary transcript is polyadenylated and contains a cap structure. (2) In the case of let-7 miRNA, a short, capped leader sequence SL1 is trans-spliced to an acceptor site at the 5´ end of the primary transcript. (3) The resulting pri-miRNA is processed by Drosha together with Pasha yielding the pre-miRNA. (4) In vertebrates the pre-miRNA is exported to the cytoplasm by Exportin-5 and (5) subsequently cleaved by Dicer. (6) The mature miRNA binds to imperfectly complementary sites in the 3´UTR of target mRNAs and represses the expression of the mRNA by a mechanism that is not completely understood.
Mode of action
MiRNAs function by binding to the 3´UTR of target genes. Although much effort has been made to elucidate the mode of action, it is remains controverial. The modes of action currently discussed include inhibition of translation either at the initiation or the elongation step, deadenylation and/or degradation of the target mRNA (Fig. 2). Among these, repression of translation initiation and transcript degradation have been observed both in cell-free lysates, in cultured cells (in vitro) and in whole animals (in vivo), suggesting that they present major and conserved mechanisms of action. GW182 proteins such as C. elegans AIN-1 and AIN-2 are required for these mechanisms, which continue to be understood in little detail.
Figure 2 miRNA mode of action
The mechanism by which miRNAs repress the target mRNA is not understood in detail. It may involve the inhibition of translation either at the initiation or the elongation step, target mRNAs deadenylation and/or degradation. Some of these repressive mechanisms may occur in so called P-bodies.
The let-7 miRNA and heterochronic phenotypes
One of the founding members of the miRNA family, let-7 is one of the best characterized miRNAs. As the name let-7 (lethal-7) implies, mutations in this miRNA are lethal. Hermaphroditic C. elegans die during the transition from larval to adult stage by bursting through the vulva. This defect is thought to be a consequence of the let-7 heterochronic phenotypes, i.e. the temporal misregulation of cell fate patterns observed in these animals.
In general, heterochronic mutations can result either in an early (precocious) or a delayed (retarded) phenotype. For instance, precocious mutants may express adult cell fates when the animal is still immature, and retarded mutants may reiterate larval cell fates in the adult stage. The heterochronic defects of let-7 mutants are best understood in a subset of epidermal cells, the seam cells. These cells normally divide at each larval stage and at the L4-to-adult transition exit the cell cycle. They subsequently fuse with the neighboring seam cells and generate the cuticular alae. In let-7 mutants the seam cell lineages are normal during the larval stages, but at the larval to adult transition, reiterate larval cell division and do not generate adult alae. The opposite, precocious phenotype, is observed in loss of function mutants of let-7 target genes (e.g. lin-41); in these cases, seam cells differentiate early during larval stages.
The fact that the let-7 system offers easily detectable phenotypes and well studied target genes, has made it a successful model to study the biogenesis of miRNAs. At the same time understanding the biogenesis and function of let-7 is an important end in itself given let-7´s roles as a major developmental regulator in C. elegans and, potentially, humans, where let-7 appears to function as a tumor suppressor gene regulating the RAS oncogene.
