2022年1月4日 · An ATP-dependent helicase has been proposed first to sep. the two siRNA strands, then the resulting single-stranded guide is thought to bind Ago2. Here, we show that Ago2 instead directly receives the double-stranded siRNA from the RISC assembly machinery. Ago2 then cleaves the siRNA passenger strand, thereby liberating the single-stranded guide.

2025年3月7日 · Ray et al. show that amyloid proteins enhance Ago2-miRNP targeting to RNA-processing bodies (PBs), which impairs miRNA activity in amyloid beta-expressing cells. This compartmentalization of Ago2 is prevented by HuR, which counteracts the effect of amyloid beta on Ago2 miRNPs by blocking its targeting to PBs.

2010年11月10日 · We find that the highly conserved tyrosine Y529, located in the small RNA 5′-end-binding pocket of Ago proteins can be phosphorylated. By substituting Y529 with a negatively charged glutamate (E) mimicking a phosphorylated tyrosine, we show that small RNA binding is strongly reduced.

2022年12月13日 · We performed experiments in HEK293T cells to determine whether mutations of AGO2 S478 (the serine corresponding to ALG-1 S642) impair miRNA function and found that, although miRNA levels were not affected upon overexpression of FLAG/hemagglutinin (HA) AGO2 S478E (Figure S6 D), FLAG/HA AGO2 S478E and the miRNA binding mutant FLAG/HA AGO2 Y529E ...

AGO2-Y529E is impaired in siRNA binding by the substitution of tyrosine to glutamic acid at amino acid 529, which is located in the binding pocket of AGO2 with the 5′-end of the siRNA guide...

Argonaute 2 (Ago2) protein is a central effector of RNA interference (RNAi) pathways and regulates mammalian genes on a global level. The mechanisms of Ago2-mediated silencing are well understood, but less is known about its regulation. Recent reports indicate that phosphorylation significantly affects Ago2 activity.

Plasmid pIRESneo-FLAG/HA-Ago2-Y529E from Dr. Kumiko Ui-Tei's lab contains the insert Human AGO2 and is published in Nucleic Acids Res. 2015 Nov 16;43 (20):9856-73. doi: 10.1093/nar/gkv1026. Epub 2015 Oct 7. This plasmid is available through Addgene.

2015年11月11日 · To investigate such possibility, we used Ago2 mutant (Ago2-Y529E), which is shown to be impaired in small RNA binding and localization in P body by the substitution of tyrosine to glutamic acid at position 529 amino acid, which is located in the binding pocket of Ago2 with 5′-end of small RNA (31,32). We purified RNAs from immunoprecipitates ...

The Ago2 Y529E mutant served as negative control since it does not bind miRNAs and should therefore not specifically associate with mRNAs. To assess mRNA binding activity, we focused on mRNAs associated with wt Ago2 and compared it to the individual mutants (Figs 8 A and EV2 ).

2014年2月27日 · Argonaute (Ago) proteins mediate posttranscriptional gene repression by binding guide miRNAs to regulate targeted RNAs. To confidently assess Ago-bound small RNAs, we adapted a mouse embryonic stem cell system to express a single epitope-tagged Ago protein family member in an inducible manner.