分子生物学全资料.ppt

Killing Messengers Double stranded RNA mediates the silencing of the translation of its corresponding mRNA This structure is characteristic of an RNase III like cleavage pattern, which led to the identification of the highly conserved Dicer family of RNase III enzymes as the mediators of dsRNA cleavage. Are there endogenous siRNAs? Interestingly, endogenously expressed siRNAs have not been found in mammals. However, the related micro(mi)RNAs have been cloned from various organisms and cell types. miRNAs are short RNA species, about 23nt in longth, which are produced by Dicer cleavage of longer, about 70nt, endogenous precursors with imperfect hairpin RNA structures. The miRNAs are believed to bind to the sites that have partial sequences complementary in the 3’ untranslated region (UTR) of their target mRNA, causing the repression of translation and the inhibition of protein synthesis. In addition to Dicer, other PAZ/PIWI domain protein (PPD) including eukaryotic translation initiation factor 2C2 (elf2C2) are likely to function in both pathways. RNAi mediated by the introduction of long dsRNA has been used as a method to investigate gene function in various organisms including plants, hydras, trypanosomes, drosophila, mosquitoes, and mouse. and FIsh? and China? The applicability of this tool in mammals is limited because the introduction of dsRNA longer than 30nt induces a sequence nonspecific INTERFERON response. Interferon triggers the degradation of mRNA by inducing 3’-5’ oligoadenylate synthase, which in turn activate RNase L. (to all mRNA species0 In addition, interferon activate the protein kinase, PKR, which phosphorylates the translation initiation factor elF2a, leading to a global inhibition of translation. dsRNAs do not work in mammals, how about siRNA directly? When chemically synthesized siRNAs were introduced co-transfectionally into fruit fly cells with luciferase reporter construct, they are functional. The results are comparable