Translational control by microRNAs

描述(由申请人提供)： 这项应用的目的是研究miRNAs在哺乳动物细胞基因表达调控中的作用。虽然在秀丽线虫、黑腹果蝇和人类中已鉴定出至少135个miRNAs，但只有两个miRNAs的功能被确定：秀丽线虫的lin-4和let-7。这两个miRNAs都通过与其靶mRNAs的3‘非编码区不完全配对来相互作用。研究表明，线虫特异的LIN-4 miRNA抑制其靶基因LIN-14的翻译。虽然已经知道高度保守的let-7的表达可以减少LIN-41基因产物在线虫中的积累，但这种抑制的机制和其他物种中let-7同源基因的靶mRNA都不清楚。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目的mRNAs的方法来分离其人靶mRNAs。在获得目的mRNAs的cDNA拷贝后，通过补骨脂素交联和定点突变来确定参与let-7/miRNA相互作用的核苷酸。为了研究let7-mRNA复合体的生理作用，我们将在培养细胞中检测含有野生型和突变的let-7结合位点的mRNAs的稳定性和翻译效率。为了了解调节mRNA翻译的miRNAs的流行程度，我们将使用cDNA微阵列在周期细胞和有丝分裂停滞细胞中寻找与多聚体相关的miRNAs。相关miRNAs的身份将通过获得cDNA拷贝和分析其序列来确定。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，并将识别在细胞周期中调节的不同的miRNA/mRNA复合体。如果miRNAs可以促进快速和可逆的翻译控制的假设是正确的，那么拟议的实验应该提供大量新的生物学信息。这个项目与健康相关的地方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.