Inorganic phosphate blocks binding of pre-miRNA to Dicer-2 via its PAZ domain.
In Drosophila, Dicer-1 produces microRNAs (miRNAs) from pre-miRNAs, whereas Dicer-2 generates small interfering RNAs from long double-stranded RNA (dsRNA), a process that requires ATP hydrolysis. We previously showed that inorganic phosphate inhibits Dicer-2 cleavage of pre-miRNAs, but not long dsRNAs. Here, we report that phosphate-dependent substrate discrimination by Dicer-2 reflects dsRNA substrate length. Efficient processing by Dicer-2 of short dsRNA 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 dsRNA 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, dsRNA. 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-miRNA 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.
Howard Hughes Medical Institute RNA Therapeutics Institute University of Massachusetts Medical School, Worcester, MA, USA.
Ryuya
R
Fukunaga
Cansu
C
Colpan
Bo W
BW
Han
Phillip D
PD
Zamore
D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine; D029721:Drosophila Proteins / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D004331:Drosophila melanogaster / Q000378:metabolism*; D005260:Female; D035683:MicroRNAs / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010710:Phosphates / Q000494:pharmacology*; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D043244:Ribonuclease III / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D029721:Drosophila Proteins; D035683:MicroRNAs; D010710:Phosphates; D012330:RNA, Double-Stranded; D011993:Recombinant Fusion Proteins; D001120:Arginine; C478069:DCR-2 protein, Drosophila; C515033:Dcr-1 protein, Drosophila; D043244:Ribonuclease III; D020365:RNA Helicases