Phosphorylation of human Argonaute proteins affects small RNA binding

• Published in: Nucleic acids research Vol. 39; no. 6; pp. 2330 - 2343
• Main Authors: Rüdel, Sabine, Wang, Yanli, Lenobel, René, Körner, Roman, Hsiao, He-Hsuan, Urlaub, Henning, Patel, Dinshaw, Meister, Gunter
• Format: Journal Article
• Language: English
• Published: England Oxford University Press 01.03.2011
• Subjects: Argonaute 2 protein; Argonaute Proteins; Data processing; Eukaryotic Initiation Factor-2 - chemistry; Eukaryotic Initiation Factor-2 - genetics; Eukaryotic Initiation Factor-2 - metabolism; Eukaryotic Initiation Factors - chemistry; Eukaryotic Initiation Factors - genetics; Eukaryotic Initiation Factors - metabolism; Gene silencing; Glutamic acid; HEK293 Cells; Humans; Mimicry; miRNA; Mutation; Phosphate; Phosphorylation; Phosphotyrosine - chemistry; Protein Binding; Protein Structure, Tertiary; Ribonuclease III - metabolism; RNA; RNA, Small Interfering - metabolism; RNA, Small Untranslated - metabolism; RNA-Binding Proteins - metabolism; siRNA; Tyrosine; Tyrosine - metabolism
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/gkq1032

Argonaute (Ago) proteins are highly conserved between species and constitute a direct-binding platform for small RNAs including short-interfering RNAs (siRNAs), microRNAs (miRNAs) and Piwi interacting RNAs (piRNAs). Small RNAs function as guides whereas Ago proteins are the actual mediators of gene silencing. Although the major steps in RNA-guided gene silencing have been elucidated, not much is known about Ago-protein regulation. Here we report a comprehensive analysis of Ago2 phosphorylation in human cells. 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. Our data suggest that a negatively charged phospho-tyrosine generates a repulsive force that prevents efficient binding of the negatively charged 5' phosphate of the small RNA.