Regulation of Argonaute slicer activity by guide RNA 3' end interactions with the N-terminal lobe.
J Biol Chem
; 288(11): 7829-7840, 2013 Mar 15.
Article
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| MEDLINE
| ID: mdl-23329841
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.
Texto completo:
1
Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Regulación de la Expresión Génica
/
Proteínas de Drosophila
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Proteínas Argonautas
Límite:
Animals
Idioma:
En
Revista:
J Biol Chem
Año:
2013
Tipo del documento:
Article