Determinate Amino Acids That Control The Localization Of Ago2 In P-Body

RNAi (RNA interference) refers to the phenomenon of specific mRNA degradation induced by highly conservative homologous dsRNA (double-stranded RNA). Ago (Argonaute) protein family includes many members of the proteins which contain the PAZ domain and PIWI domain. In recent years, it was found that many members of Ago protein family played an important role in RNAi. Ago2 (Argonaute 2), as Ago protein family members, was found at first in the fly. In the cells of the fly and human, Ago2 is the core protein of RNAi machine RSIC (RNA-induced silencing complex) and plays the key role in the RNAi.P-body (Cytoplasmic processing body) was found at first in yeast and associated with the mRNA decay. Many proteins, which function in the transcriptional regulation, RNA degradation and translational repression, were colocalized in P-Bodies. Now, the sufficient evidences show that RNAi system and the P-body formation was linked. For example, Ago2 was gathered within the P-bodies. The combination of Ago2 with mRNA will affect the presence of P-Body in cells, and so on. Because Ago2 and P-Body were closely linked with the RNAi, study on Ago2 localization in P-Body has important significance for the understanding the relationship between P-Body and RNAi and also the mechanism of RNAi.We obtained by chance an Ago2 mutant (Ago2-G32W-F128L-R196Q -P458S-T741A-S752G), which contains six amino acid mutation and was completely out of the P-Bodies. In this thesis, we will address the key amino acids which impact on the localization of Ago2 in P-Bodies through construction of a series of mutants of Ago2 and utilization of cellular immunofluorescence staining. The result shows that Single point mutants (except S752G) and multiple site mutants(not containing S752G) do not affect the localization of Ago2 in P-Bodies. Compared to the other five amino acids, the 752 Ser of Ago2 is particularly important, only the S752G mutation reduced significantly the localization of Ago2 in P-Bodies. But the S752G mutation alone is not enough to make Ago2 completely detached from the P-Body, it also needs the assistance of 32Gly 128Phe, 458Pro mutation. Ago2 mutant (G32W-F128L-P458S-S752G) don't localize in P-Body. This shows that these amino acids control the localization of Ago2 in the P-Body.