miRNa靶标预测及验证课件.pptVIP

miRNA 靶标预测和功能验证 miRNA 的靶标预测 Seeds of miRNA Target Prediction UTR Context Target-Prediction Tools 大量的miRNA 被发现，产生了许多的问题，这个非编码的小RNA 在细胞中的作用是什么？ 回答这个问题的关键是要找到miRNA 所调节的基因。在植物中靶标基因与miRNA是互补很容易预测到，但是在动物中只是偶尔才发现。 现在预测的方法有很多但是有三个结论是实验验证过的。 The first major conclusion is that requiring conserved Watson–Crick pairing to the 5′ region of the miRNA centered on nucleotides 2–7, which is called the miRNA “seed” The second conclusion is that conserved pairing to the seed region can also be sufficient on its own for predicting conserved targets above the noise of false-positive predictions The third major conclusion: highly conserved miRNAs have very many conserved targets miRNA 与靶标基因集合位点的三种类型 规范型 3’- supplement site 3’-compensatory site UTR Context positioning within the 3’UTR at least 15 nt from the stop codon positioning away from the center of long UTRs AU-rich nucleotide composition near the site or other measures of site accessibility proximity to sites for coexpressed miRNAs. Target-Prediction Tools 并不是只调节3’UTR 3’UTRORF5’UTR 功能验证 miRNA/mRNA interactions 在不表达miRNA的细胞中异源表达miRNA和含有mRNA 3’UTR的质粒。 含有mRNA 3’UTR的质粒转入表达miRNA的细胞中，同时做突变质粒的对照。 miRNA and target mRNA co-expression 用实时定量检测miRNA 和mRNA 的表达。 miRNA effect on target protein 过量表达miRNA 对mRNA翻译的影响，从蛋白水平检测。 抑制miRNA的表达，检测对蛋白表达的影响。 miRNA effects on target biological function 主要是包括信号通路，细胞周期，细胞分化，细胞凋亡，细胞迁移 * *