Phosphorylation of human Argonaute proteins affects small RNA binding.
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.
Center for integrated protein science Munich, Laboratory of RNA Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, 82152 Martinsried, Germany.
Sabine
S
Rüdel
Yanli
Y
Wang
René
R
Lenobel
Roman
R
Körner
He-Hsuan
HH
Hsiao
Henning
H
Urlaub
Dinshaw
D
Patel
Gunter
G
Meister
D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D009154:Mutation; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D034741:RNA, Small Interfering / Q000378:metabolism; D058727:RNA, Small Untranslated / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism; D043244:Ribonuclease III / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
C115848:AGO1 protein, human; C466422:AGO2 protein, human; D060565:Argonaute Proteins; D015852:Eukaryotic Initiation Factor-2; D039642:Eukaryotic Initiation Factors; D034741:RNA, Small Interfering; D058727:RNA, Small Untranslated; D016601:RNA-Binding Proteins; C542384:TNRC6B protein, human; D019000:Phosphotyrosine; D014443:Tyrosine; D043244:Ribonuclease III