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核苷酸小RNA，被认为控制着 转录后水平的基因表达。尽管miRNA介导的基因的重要性 现在调控是显而易见的，因为miRNA基因被证明在动物中扮演着不同的功能角色，他们的 行动机制仍然难以捉摸。我们最近发现，编码相同的miRNA基因成熟 MiRNA可能具有不同的生物学活性，这些活性由它们的同源前miRNA环决定， 揭示了在miRNA前环路中编码的意想不到的调控复杂性。这些发现促使我们 重新检查miRNA领域的一些基本假设。它被记录在最初的发现中 线虫lin-4基因的突变和缺失总是会影响包括初级 (PrI)、前体(Pre)和成熟的miRNA物种，它们都包含与同源序列互补的序列 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 仅有成熟的miRNA物种。总之，当前假设的局限性和我们令人惊讶的发现 前miRNA环功能提示Pri-和前miRNA物种可能在靶基因中发挥直接作用 监管。在这里，我们建议研究前miRNA环控制miRNA活性的机制。 使用生化和遗传方法的基因。我们将进一步剖析结构和序列元素 除了对miRNA基因的活性很重要的成熟miRNA区域(目标1)之外，识别蛋白质 和有助于前miRNA环功能的RNA组分(目标2)，并描述PremiRNA的作用 靶基因识别中的环核苷酸(目标3)。这项拟议的研究将提供基本的见解 通过miRNA基因进行基因调控，并产生用于靶基因预测和设计的新原理 更有效的针对信使RNA和miRNA基因的基因沉默分子。

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.