title: Regulation of Argonaute slicer activity by guide RNA 3' end interactions with the N-terminal lobe.
abstract: Structural studies indicate that binding of both the guide RNA (siRNA and miRNA) and the target mRNA trigger substantial conformational changes in the Argonaute proteins. Here we explore the role of the N-terminal lobe (and its PAZ domain) in these conformational changes using biochemical and cell culture-based approaches. In vitro, whereas deletion (or mutation) of the N-terminal lobe of DmAgo1 and DmAgo2 had no effect on binding affinity to guide RNAs, we observed a loss of protection of the 3' end of the guide RNA and decreased target RNA binding; consistent with this, in cells, loss of function DmAgo1 PAZ variant proteins (PAZ6 and ΔN-PAZ) still bind RNA, although the RNAs are shorter than normal. We also find that deletion of the N-terminal lobe results in constitutive activation of endogenous PIWI domain-based cleavage activity in vitro, providing insights into how cleavage activity may be regulated in vivo in response to different types of pairing interactions with the target mRNAs.
authors: Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors: Junho K
authors: JK
authors: Hur
authors: Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors: Michelle K
authors: MK
authors: Zinchenko
authors: Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors: Sergej
authors: S
authors: Djuranovic
authors: Howard Hughes Medical Institute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205. Electronic address: ragreen@jhmi.edu.
authors: Rachel
authors: R
authors: Green
mesh_terms: D000818:Animals; D060565:Argonaute Proteins / Q000096:biosynthesis / Q000378:metabolism*; D029721:Drosophila Proteins / Q000096:biosynthesis*; D004331:Drosophila melanogaster; D005786:Gene Expression Regulation; D007313:Insecta; D035683:MicroRNAs / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism; D058727:RNA, Small Untranslated
chemical_list: C489654:AGO1 protein, Drosophila; C433807:AGO2 protein, Drosophila; D060565:Argonaute Proteins; D029721:Drosophila Proteins; D035683:MicroRNAs; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D058727:RNA, Small Untranslated