Argonaute proteins are universal in post-transcriptional regulation by small interfering RNAs (siRNAs) and micro RNAs (miRNAs). siRNAs and miRNAs are 21∼23 nt RNAs that bind to target mRNA through base-paring for the regulatory process. In RNA interference (RNAi), siRNA associated Argonaute becomes part of RNA-induced silencing complex (RISC) and mediates enzymatic cleavage of target mRNA. miRNA associated Argonaute and cofactors from a miRNA ribonucleoprotein complex (miRNP) and induces cleavage-independent translational repression through mechanisms still incompletely understood. In order to understand miRNA-mediated regulation, we investigated Drosophila melanogaster Argonaute 1 (DmAgol), which mediates repression in miRNP in flies. By using bioinformatic, in vitro, and cell-based assays, we identified the MID domain of DmAgol as allosteric regulator of protein function in miRNA-mediated repression. Mutation analyses revealed molecular features involved in activation of Argonaute, upon binding of 5' end of miRNA to the MID domain, to repress target mRNA. We further show that the protein activation is also linked to association with GW182, which is a downstream gene in miRNA-mediated repression pathway.
Molecular structures of siRNA and miRNAs are similar. and certain Argonautes are capable of binding to both classes of small RNA to regulate target mRNA in RNAi or translational repression. siRNA binds to target mRNAs with perfect base-paring, while miRNAs generally interact with mRNA in the 3' untranslated region (UTR) through incomplete complementarity. However, how Argonautes recognize the differences and decide siRNA or miRNA pathway for repression is not completely understood. Structural studies suggested that small RNAs bind to Argonaute with the 3' end anchored in the PAZ domain of Argonaute, and the interaction can be lost depending on binding of target mRNA. To study the role of the PAZ domain, we conducted biochemical and cell-culture based assays on protein-RNA interactions of Drosophila Argonautes. Our results indicate that the PAZ domain does not contribute to small RNA binding affinity, but protects bound small RNA from degradation. We also show that the PAZ domain facilitates target mRNA association. Most importantly, we find that loss of the PAZ domain leads to constitutive activation of endoribonuclease function of Argonaute. Our results also showed that if miRNA-target mRNA is bound to Argonaute, the interactions between the PAZ domain and the 3' terminus of miRNA subdue cleavage activity of Argonaute protein.
The work presented in the thesis provides insights that Argonautes conformations are altered by protein-RNA interactions, and the changes regulate Argonaute functions in repression of target mRNA through RNAi and miRNA pathways.
|School:||The Johns Hopkins University|
|School Location:||United States -- Maryland|
|Source:||DAI-B 74/01(E), Dissertation Abstracts International|
|Keywords:||RNA interference, Translational repression, miRNA|
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