Coordinated activities of human dicer domains in regulatory RNA processing.
The conserved ribonuclease Dicer generates microRNAs and short-interfering RNAs that guide gene silencing in eukaryotes. The specific contributions of human Dicer's structural domains to RNA product length and substrate preference are incompletely understood, due in part to the difficulties of Dicer purification. Here, we show that active forms of human Dicer can be assembled from recombinant polypeptides expressed in bacteria. Using this system, we find that three distinct modes of RNA recognition give rise to Dicer's fidelity and product length specificity. The first involves anchoring one end of a double-stranded RNA helix within the PAZ domain, which can assemble in trans with Dicer's catalytic domains to reconstitute an accurate but non-substrate-selective dicing activity. The second entails nonspecific RNA binding by the double-stranded RNA binding domain, an interaction that is essential for substrate recruitment in the absence of the PAZ domain. The third mode of recognition involves hairpin RNA loop recognition by the helicase domain, which ensures efficient processing of specific substrates. These results reveal distinct interactions of each Dicer domain with different RNA structural features and provide a facile system for investigating the molecular mechanisms of human microRNA biogenesis.
Department of Molecular and Cell Biology, University of California, Berkeley, CA 94720, USA.
Enbo
E
Ma
Kaihong
K
Zhou
Mary Anne
MA
Kidwell
Jennifer A
JA
Doudna
D000251:Adenosine Triphosphatases / Q000235:genetics / Q000378:metabolism; D001419:Bacteria / Q000235:genetics; D053487:DEAD-box RNA Helicases / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D035683:MicroRNAs / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020365:RNA Helicases / Q000235:genetics / Q000378:metabolism; D012330:RNA, Double-Stranded / Q000235:genetics* / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D043244:Ribonuclease III / Q000235:genetics* / Q000378:metabolism*; D013379:Substrate Specificity
D035683:MicroRNAs; D010446:Peptide Fragments; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; D011994:Recombinant Proteins; C408087:DICER1 protein, human; D043244:Ribonuclease III; D000251:Adenosine Triphosphatases; D053487:DEAD-box RNA Helicases; D020365:RNA Helicases