Translational control by microRNAs

描述（由申请人提供）： 本申请的目的是研究miRNAs在哺乳动物细胞基因表达调控中的作用。尽管在秀丽隐杆线虫、黑腹果蝇和人类中已经鉴定出至少135种miRNAs，但仅确定了两种miRNAs的功能作用：C.优雅这两种miRNAs通过与其靶mRNA的3'非编码区不完全配对而相互作用。结果表明，C.线虫特异性lin-4 miRNA抑制其靶mRNA lin-14的翻译。尽管已知高度保守的let-7的表达减少lin-41基因产物在C.在线虫中，这种抑制的机制和let-7直向同源物在其他物种中的靶mRNA都是未知的。为了研究let-7在人类细胞中的功能，我们将通过在细胞中表达生物素标记的let-7 RNA并通过亲和层析分离靶mRNA来分离其人类靶mRNA。在获得靶mRNA的cDNA拷贝后，将通过peptide交联和定点诱变来确定参与let-7/miRNA相互作用的核苷酸。为了研究let 7-mRNA复合物的生理作用，将在培养的细胞中检查含有野生型和突变的let-7结合位点的mRNA的稳定性和翻译效率。为了了解调节mRNA翻译的miRNA的流行情况，我们将使用cDNA微阵列寻找与周期细胞和有丝分裂停滞细胞中的多聚体相关的miRNA。通过获得cDNA拷贝并分析其序列来确定相关miRNA的身份。使用几种不同的方法，本提案中描述的实验将解决高度保守的miRNA let-7的功能，并将鉴定在细胞周期中受调控的不同miRNA/mRNA复合物。如果miRNAs能够促进快速和可逆的翻译调控这一假设是正确的，那么这些实验将提供大量新的生物学信息。本项目的健康相关性在于这种新的基因调控机制在细胞生长或发育中的潜在作用。

DESCRIPTION (provided by applicant): The goal of this application is to investigate the roles of miRNAs in the regulation of gene expression in mammalian cells. Although at least 135 miRNAs have been identified in Caenorhabditis elegans, Drosophila melanogaster and humans, a functional role has been determined for only two: lin-4 and let-7 of C. elegans. Both of these miRNAs interact by incomplete pairing with the 3' noncoding regions of their target mRNAs. It was demonstrated that the C. elegans-specific lin-4 miRNA inhibits translation of its target mRNA, lin-14. Although the expression of the highly conserved let-7 is known to reduce the accumulation of the lin-41 gene product in C. elegans, neither the mechanism of this inhibition nor the target mRNA of let-7 orthologues in other species are known. To investigate the function of let-7 in human cells, we will isolate its human target mRNAs by expressing biotin-tagged let-7 RNA in cells and isolating target mRNAs by affinity chromatography. After obtaining cDNA copies of the target mRNAs, the nucleotides that participate in let-7/miRNA interactions will be determined by psoralen crosslinking and site-directed mutagenesis. To study the physiological roles of let7-mRNA complexes, the stability and translational efficiency of mRNAs containing wildtype and mutated binding sites for let-7 will be examined in cultured cells. To gain an understanding of the prevalence of miRNAs that regulate mRNA translation, we will search for miRNAs that are associated with polysomes in cycling cells and in mitotically arrested cells using cDNA microarrays. Identities of associated miRNAs will be determined by obtaining cDNA copies and analyzing their sequence. Using several different approaches, the experiments described in this proposal will address the function of the highly conserved miRNA let-7 and will identify distinct miRNA/mRNA complexes that are regulated during the cell cycle. If the hypothesis is correct that miRNAs can facilitate a rapid and reversible control of translation, then the proposed experiments should provide a large amount of novel biological information.The health-relatedness of this project lies in the potential roles of this novel mechanism of gene regulation in cell growth or development.