MicroRNAs : principles of target recognition and developmental roles

Abstract

MicroRNAs (miRNAs) are ~21-24 nt non-coding RNAs that mediate the degradation and translational repression of target mRNAs. The genomes of vertebrate organisms encode hundreds of miRNAs, each of which may regulate hundreds of mRNA targets. Thus, miRNAs are crucial post-transcriptional regulators engaged in vast regulatory networks. To date, the characteristics of these networks remain mysterious due to the difficulty of identifying miRNA targets through either experimental or computational means. To understand the physiological roles of miRNAs in animal species, it is of fundamental importance to elucidate the structure of the targeting networks in which they participate. The recognition of a miRNA target is guided largely by perfect Watson-Crick base pairing interactions between nucleotides 2-7 from the 5' end of the miRNA (i.e., the "seed" region) and complementary motifs embedded in the 3' UTRs of the target mRNAs. The prevalence of these motifs throughout the transcriptome poses a challenge to our understanding of how specificity emerges: since the presence of a motif is not sufficient to mediate target repression, what contextual features discriminate effective target sites from ineffective ones? Further complicating this is the proposition that "noncanonical" sites lacking perfect seed pairing might mediate repression, which would expand the potential number of functional target sites by orders of magnitude. In the second chapter of this work, we define the features that predict effective miRNA target sites, incorporating their relative influence into a quantitative model which can outperform existing computational models and experimental approaches in target identification. Though the molecular roles of miRNAs in gene regulation have long been appreciated, the functions of most miRNAs in living organisms has remained elusive. In the third chapter of this work, we discuss the consequences of genetic ablation of miR-196, a deeply conserved miRNA that is predicted to simultaneously repress many HOX genes, in the mouse. We propose a role for miR-196 in the spatial patterning of the vertebrate axial skeleton. Isolating the cell populations that express the miRNA during early mammalian development, we attempt to characterize the direct in vivo targets of miR-196 and dissect the molecular underpinnings of the phenotypes observed.