基因表达调控 第三课 mRNA水平研究9-29.ppt

（七）差异表达基因筛选方法 表达序列标签(expressed sequencing tag，EST) 技术，1991 差异显示RT-PCR(different display reverse transcription PCR，DDRT-PCR)，1992 抑制性差减杂交(suppression subtractive hybridisation，SSH)，1996 基因芯片(gene chip or micrOarray) 基因表达的系列分析(serial analysis of gene expression，SAGE)，1995 基于高通量测序的数字基因表达谱(DGE)分析 转录抑制剂： actinomycin D (ActD), 放线菌素D 5,6-dichloro-1–D-ribofuranosyl-benzimidazole (DRB), α-amanitin (α-Am) （八）mRNA稳定性分析 荧光素酶报告基因分析系统 二、mRNA稳定性调节的机制 AU-rich elements (AREs) : AUUUA, 7-9% of cellular mRNAs These elements are recognized by trans-acting factors, such as mRNA-binding proteins that bind directly or indirectly to the cis-acting elements and promote the deadenylation and degradation of the mRNA. AU-rich element RNA-binding protein 1 (AUF1), tristetraprolin (TTP) , KH-type splicing regulatory protein (KSRP) embryonic lethal abnormal vision (ELAV)-like protein 1 (HuR) and polyadenylate-binding protein-interacting protein 2 (PAIP2) （1）AU-rich sequences and mRNA binding proteins （2）Regulatory non-coding RNA miRNAs act by pairing to the mRNAs of protein-coding genes to direct their repression. lncRNAs show different mechanism of action, varying from chromatin remodeling, transcriptional responses and RNA processing. （3）Alternative polyadenylation (APA) APA consist of two steps, cleavage and polyadenylation of RNAs, which are maturation events that cut and add an oligo(dA) tail to the 3’ end of the nascent transcript. This processing protects mRNAs from degradation and increases their stability. The specific cleavage position and the efficiency of the process depend on the interaction between trans-acting polyadenylation factors and cis-elements present in the pre-mRNAs, such as the central sequence motif AAUAAA. Most mRNA regulatory elements are situated within the 5’ and 3’untranslated regions (UTRs). The mRNA stability is regulated by the complex interplay of cis-acting equences within the 3’UTR of the mRNA and trans-acting factors, such as RNA binding protein