Identification and Functional Characterization of Zebrafish microRNAs

描述（由申请人提供）：多个真核生物测序项目已经导致了令人惊讶的发现，即蛋白质编码基因的数量远远少于基于总蛋白质含量预测的数量。这强调了这样一个事实，即基因表达的调控对于使相对有限数量的基因能够编码组织和发育特异性模式中的广泛蛋白质同种型至关重要。在过去的几年里，很明显，只关注蛋白质编码基因忽略了非编码RNA的基因，这些基因似乎占基因总数的1%以上。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包括一个被称为microRNA（miRNA）的基因家族。miRNA通过降解靶mRNA或介导翻译抑制以序列特异性方式调节基因表达。在这个建议中，我们试图在全球范围内确定所有的斑马鱼miRNAs，并确定其细胞，组织和发育特异性表达模式，使用一种新的微阵列策略。真核细胞编码大约250-300种miRNA，但这些RNA的靶点几乎完全未知。使用阵列数据，我们将识别miRNA靶点，并检查miRNA功能获得和功能丧失的表型后果。miRNAs最近才被鉴定的事实说明了使用正向遗传筛选鉴定所有基因的困难，特别是小的脊椎动物基因，如编码miRNAs的基因。据估计，10%或更多的真核基因可能由一个或多个miRNA调控，本提案的目标是使用斑马鱼系统来回答有关miRNA如何调控早期发育的问题。由于miRNAs是高度保守的，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.