The Role of Pre-miRNA Loop in Target Regulation by microRNA Genes

摘要 microRNA（miRNA）基因编码一类丰富的~22个核苷酸的小RNA，其被认为控制 转录后水平的基因表达。虽然miRNA介导的基因的重要性 调控现在是显而易见的，因为miRNA基因被证明在动物中起着不同的功能作用， 作用机制仍然难以捉摸。我们最近发现编码相同成熟RNA的miRNA基因， miRNA可能具有不同的生物活性，这些生物活性由它们的同源前体miRNA环决定， 揭示了前体miRNA环中编码的意想不到的调控复杂性。这些发现促使我们 重新审视miRNA领域的一些基本假设。在最初的发现中， 梭线虫lin-4基因lin-4基因中的突变和缺失总是影响包括主要的 (pri)前体（前）和成熟的miRNA种类，它们都含有与同源物互补的序列。 靶基因因此，在蠕虫和小鼠中观察到的miRNA基因丢失的表型不能归因于 成熟的miRNA种类。总之，当前假设的局限性和我们对 pre-miRNA环的功能表明，pri-miRNA和pre-miRNA种类可能在靶基因中发挥直接作用， 调控在这里，我们打算研究前体miRNA环控制miRNA活性的机制， 利用生物化学和遗传学方法研究基因。我们将进一步剖析结构和序列元素 除了对miRNA基因活性重要的成熟miRNA区域（目的1）， 和RNA成分，有助于前体miRNA环功能（目的2），并描绘premiRNA的作用 环核苷酸在靶基因识别中的作用（aim 3）。这项拟议中的研究将揭示基本的见解， 研究miRNA基因的基因调控，并为靶基因预测和设计 更有效的基因沉默分子靶向信使RNA和miRNA基因。

ABSTRACT MicroRNA (miRNA) genes encode an abundant class of ~22-nucleotide small RNAs that are thought to control gene expression at the post-transcriptional levels. Although the importance of miRNA-mediated gene regulation is now evident, as miRNA genes were shown to play diverse functional roles in animals, their mechanisms of action remain elusive. We have recently found that miRNA genes encoding identical mature miRNAs could have distinct biological activities that are determined by their cognate pre-miRNA loops, revealing unexpected regulatory complexity encoded in the pre-miRNA loops. These findings prompted us to reexamine some of the fundamental assumptions in the miRNA field. It was noted in the original discovery of the C. elegans lin-4 gene that mutations and deletions in lin-4 genes invariably affect including the primary (pri), precursor (pre), and mature miRNA species, which all contain sequences complementary to cognate target genes. Thus, phenotypes observed for loss of miRNA genes in worms and mice cannot be attributed to mature miRNA specie alone. Together, the limitations of current assumption and our surprising discovery of pre-miRNA loop function suggest that pri- and pre-miRNA species may play direct roles in target gene regulation. Here we propose to examine the mechanisms by which pre-miRNA loops control activity of miRNA genes using biochemical and genetic approaches. We will further dissect the structure and sequence elements beyond mature miRNA regions that are important for the activity of miRNA genes (aim 1), identify the protein and RNA components that contribute to the pre-miRNA loop functions (aim 2), and delineate the roles of premiRNA loop nucleotides in target gene recognition (aim 3). The proposed study will shed fundamental insights into gene regulation by miRNA genes and yield novel principles for target gene prediction and for designing more efficient gene silencing molecules targeting messenger RNAs and miRNA genes.