An evolutionary perspective on the sequence, mechanism, and regulatory function of animal microRNAs

Abstract

(cont.) The vertebrate-specific miR-196 family is encoded at three paralogous loci in the mammalian Hox clusters, and has complementarity to messages of several Hox genes, including Hox8 paralogues. RNA fragments diagnostic of miR-196-directed cleavage of Hoxb8 were detected in mouse embryos. Cell culture experiments demonstrated down-regulation of Hoxb8, Hoxc8, Hoxd8, and Hoxa7 and supported a cleavage mechanism for miR-196-directed repression of Hoxb8. These results point to a miRNA-mediated mechanism for the posttranscriptional restriction of Hox gene expression during vertebrate development and demonstrate that metazoan miRNAs can repress expression of their natural targets through mRNA cleavage in addition to inhibiting a translational step. Inhibition of the two Hox miRNAs in chick embryos, resulted in axial skeletal patterning defects in domains that overlap considerably with Hox target and miRNA expression, supporting a specialization of miRNAs in Hox gene regulation, and consistent with action by miR-196 and miR-10 to refine posterior boundaries at relative levels of expression for multiple Hox genes. The genomic distribution of target sites and Hox patterns of expression suggest that the miRNAs further act in concert with more posteriorly expressed Hox genes to impose a functional hierarchy over more anterior ones, a molecular mechanism consistent with 'posterior prevalence'. The posttranscriptional downregulation of more 3' and anteriorly expressed Hox genes by miR-196 constitutes an evolutionarily recent regulatory layer of the highly constrained Hox network, one which recapitulates modes of interactions existing at multiple levels of gene expression.

Noncoding RNAs are encoded by diverse genomes and play many functional roles. MicroRNAs (miRNAs) are endogenous -22-nucleotide noncoding RNAs, derived from larger hairpin precursors, which act by base-pairing to mRNAs to target these transcripts for destruction or translational repression. miRNA genes have been discovered in viral and multicellular genomes. The computational procedure MiRscan was developed to identify miRNA genes conserved in more than one genome and applied to the identification of vertebrate miRNAs. Starting with conserved mouse and human sequences with potential for hairpin formation, and subsequent comparison to fish, 15,000 human genomic loci were identified within aligned regions outside protein coding genes, and ranked according to criteria based on shared features of a training set of the first 50 experimentally verified C. elegans miRNAs. 188 high-scoring candidates, including 74% of human miRNAs known in 2002, were further examined. Fourteen candidate miRNAs were close paralogues of known miRNAs, and 38 candidates were experimentally validated using cDNA libraries of small RNAs made from zebrafish. Of the 38 verified miRNAs, 21 were sequences identified by random cloning and sequencing of cDNA libraries, while 17 were found by applying a directed PCR approach to the same libraries. The miR-196 and miR-10 families are transcribed from genomic loci within clusters of Hox transcription factor genes, and in turn mediate the posttranscriptional repression of neighbouring Hox transcripts, with conserved and extensive targeting of Hox genes located in paralogous groups that are 3' but not 5' of each miRNA locus, relative to the direction of transcription within a given cluster.