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Inorganic phosphate blocks binding of pre‐mi RNA to Dicer‐2 via its PAZ domain
Author(s) -
Fukunaga Ryuya,
Colpan Cansu,
Han Bo W,
Zamore Phillip D
Publication year - 2014
Publication title -
the embo journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 7.484
H-Index - 392
eISSN - 1460-2075
pISSN - 0261-4189
DOI - 10.1002/embj.201387176
Subject(s) - dicer , ribonuclease iii , biology , rna silencing , cleavage (geology) , microrna , biochemistry , microbiology and biotechnology , argonaute , rna , small interfering rna , rna interference , gene , paleontology , fracture (geology)
In Drosophila , Dicer‐1 produces micro RNA s (mi RNA s) from pre‐mi RNA s, whereas Dicer‐2 generates small interfering RNA s from long double‐stranded RNA (ds RNA ), a process that requires ATP hydrolysis. We previously showed that inorganic phosphate inhibits Dicer‐2 cleavage of pre‐mi RNA s, but not long ds RNA s. Here, we report that phosphate‐dependent substrate discrimination by Dicer‐2 reflects ds RNA substrate length. Efficient processing by Dicer‐2 of short ds RNA requires a 5′ terminal phosphate and a two‐nucleotide, 3′ overhang, but does not require ATP . Phosphate inhibits cleavage of such short substrates. In contrast, cleavage of longer ds RNA requires ATP but no specific end structure: phosphate does not inhibit cleavage of these substrates. Mutation of a pair of conserved arginine residues in the Dicer‐2 PAZ domain blocked cleavage of short, but not long, ds RNA . We propose that inorganic phosphate occupies a PAZ domain pocket required to bind the 5′ terminal phosphate of short substrates, blocking their use and restricting pre‐mi RNA processing in flies to Dicer‐1. Our study helps explain how a small molecule can alter the substrate specificity of a nucleic acid processing enzyme.