Multiple eukaryotic sequencing projects have led to the surprising finding that the number of protein coding genes is far less than that predicted based on total protein content. This underscores the fact that regulation of gene expression is crucial to enable a relatively limited number of genes to encode a wide array of protein isoforms in a tissue- and developmental-specific pattern. Within the last couple of years, it has become apparent that focusing on just protein coding genes ignores genes for noncoding RNAs which appear to make up more than 1% of the total number of genes. Several classes of RNA are well known (tRNA, mRNA, and rRNA) but perhaps the most interesting class of noncoding RNAs includes a family of genes referred to as microRNAs (miRNAs). miRNAs function to regulate gene expression in a sequence specific manner by either degrading target mRNAs or by mediating translational repression. In this proposal, we seek to globally identify all zebrafish miRNAs and determine their cell-, tissue-, and developmental-specific expression patterns using a novel microarray strategy. Eukaryotic cells encode approximately 250-300 miRNAs yet the targets of these RNAs are almost entirely unknown. Using the array data, we will identify miRNA targets and examine the phenotypic consequences of miRNA gain-of-function and loss-of-function. The fact that miRNAs have only recently been identified illustrates the difficulty of identifying all genes using forward genetic screens, especially small vertebrate genes such as those encoding miRNAs. It has been estimated that 10% or more of eukaryotic genes might be regulated by one or more miRNAs and the goal of this proposal is to use the zebrafish system to answer questions about how miRNAs regulate early development. Because miRNAs are highly conserved, it seems certain that information gleaned using zebrafish will be directly applicable to humans and uncover potential roles for human miRNAs in normal cell function, development, and disease.
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