Identification and Functional Characterization of Zebrafish microRNAs

描述(申请人提供)：多个真核测序项目导致了一个令人惊讶的发现，蛋白质编码基因的数量远远少于基于总蛋白质含量的预测。这突显了这样一个事实，即对基因表达的调控对于使相对有限的基因能够以组织和发育特有的模式编码广泛的蛋白质异构体至关重要。在过去的几年里，很明显，只关注蛋白质编码基因而忽略了非编码RNA的基因，而非编码RNA似乎占基因总数的1%以上。有几类RNA是众所周知的(tRNA、mRNA和rRNA)，但也许最有趣的一类非编码RNA包括一组被称为microRNAs(MiRNAs)的基因。MiRNAs通过降解靶mRNAs或介导翻译抑制，以序列特异性的方式调节基因的表达。在这项建议中，我们寻求全球识别所有斑马鱼miRNAs，并使用新的微阵列策略确定它们在细胞、组织和发育中的特异表达模式。真核细胞编码大约250-300个miRNAs，但这些RNAs的靶标几乎完全未知。使用阵列数据，我们将识别miRNA靶标，并检查miRNA功能增益和功能丧失的表型后果。最近才发现miRNAs的事实说明了使用正向遗传筛选识别所有基因的难度，特别是像编码miRNAs的小脊椎动物基因。据估计，10%或更多的真核基因可能受到一个或多个miRNAs的调控，本提案的目的是利用斑马鱼系统来回答有关miRNAs如何调控早期发育的问题。由于miRNAs高度保守，似乎可以肯定的是，使用斑马鱼收集的信息将直接适用于人类，并揭示人类miRNAs在正常细胞功能、发育和疾病中的潜在作用。

DESCRIPTION (provided by applicant): 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.