TY - JOUR
T1 - Phosphate-binding pocket in Dicer-2 PAZ domain for high-fidelity siRNA production
AU - Kandasamy, Suresh K.
AU - Fukunaga, Ryuya
N1 - Funding Information:
We are grateful to Dr. Richard Carthew (Northwestern University) for fly strains of white-inverted repeat, dicer-2null, and dicer-2G31R; Dr. Bo Han (Pacific Biosciences) and Dr. Junko Tsuji (University of Massachusetts Medical School) for their help in the bioinformatics analysis; and Susan Liao and Dr. Li Zhu, members of R.F. laboratory, for their comments on the manuscript. This work was supported by Grant 15SDG23220028 from the American Heart Association and funds provided by the Department of Biological Chemistry at the Johns Hopkins School of Medicine (to R.F.).
PY - 2016/12/6
Y1 - 2016/12/6
N2 - The enzyme Dicer produces small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22-24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5'-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5'-monophosphate of a long dsRNA substrate is anchored by the phosphatebinding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.
AB - The enzyme Dicer produces small silencing RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs). In Drosophila, Dicer-1 produces ∼22-24-nt miRNAs from pre-miRNAs, whereas Dicer-2 makes 21-nt siRNAs from long double-stranded RNAs (dsRNAs). How Dicer-2 precisely makes 21-nt siRNAs with a remarkably high fidelity is unknown. Here we report that recognition of the 5'-monophosphate of a long dsRNA substrate by a phosphate-binding pocket in the Dicer-2 PAZ (Piwi, Argonaute, and Zwille/Pinhead) domain is crucial for the length fidelity, but not the efficiency, in 21-nt siRNA production. Loss of the length fidelity, meaning increased length heterogeneity of siRNAs, caused by point mutations in the phosphate-binding pocket of the Dicer-2 PAZ domain decreased RNA silencing activity in vivo, showing the importance of the high fidelity to make 21-nt siRNAs. We propose that the 5'-monophosphate of a long dsRNA substrate is anchored by the phosphatebinding pocket in the Dicer-2 PAZ domain and the distance between the pocket and the RNA cleavage active site in the RNaseIII domain corresponds to the 21-nt pitch in the A-form duplex of a long dsRNA substrate, resulting in high-fidelity 21-nt siRNA production. This study sheds light on the molecular mechanism by which Dicer-2 produces 21-nt siRNAs with a remarkably high fidelity for efficient RNA silencing.
KW - Dicer
KW - DsRNA
KW - Phosphate
KW - RNA silencing
KW - SiRNA
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U2 - 10.1073/pnas.1612393113
DO - 10.1073/pnas.1612393113
M3 - Article
C2 - 27872309
AN - SCOPUS:85003038388
SN - 0027-8424
VL - 113
SP - 14031
EP - 14036
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 49
ER -