MicroRNA cloning and bioinformatic analysis

Abstract

Part I. Two gene-regulatory noncoding RNAs (ncRNAs), let-7 RNA and lin-4 RNA, were previously discovered in the C. elegans genome. The let-7 gene is conserved across a wide range of genomes, suggesting that these ncRNAs represent a wider class of gene-regulatory RNAs. Both lin-4 and let-7 RNAs are generated from stem-loop precursor RNAs, and share a common biochemical signature, namely 5'-terminal phosphate and 3'-terminal hydroxyl groups. We refer to ncRNAs that share the characteristic size, biochemical signature, and precursor structures of let-7 and lin-4 as microRNAs (miRNAs). The size of this class of genes, and its prevalence in other genomes, are unknown. Therefore, we developed an experimental and bioinformatics strategy to identify novel miRNA genes. We discovered a total of 75 miRNA genes in the C. elegans genome, and orthologues for a majority of these were computationally identified in the C. briggsae, D. melanogaster or H. sapiens genomes. Northern analysis was used to confirm and analyze the expression of these miRNAs. The data set has implications for understanding miRNA gene regulation, miRNA processing, and regulation of miRNA genes. Part II. Directed molecular evolution has previously been applied to generate RNAs with novel structures and functions. This method works because nucleic acids can be selected, randomized, amplified and characterized using polymerase chain reaction (PCR)-based methods. Here we present a novel method for extending directed molecular evolution to the realm of peptide selections by linking a peptide to its encoding mRNA.

(cont.) A proof of principle selection for two different peptides indicates that this tRNA should prove useful in discovering more complex protein molecules using directed molecular evolution.