Abstract
Dicer is a central enzyme for processing small RNAs in RNA interference. It is able to cleave both hairpin and long double-stranded RNA precursors into microRNAs and small interfering RNAs respectively. While humans have one Dicer that is able to process both substrates, many organisms have multiple copies. Understanding how organisms with two Dicers chose specific RNAs for cleavage could give insight into how the human protein functions. We have turned to the thermophilic fungus Sporotrichum thermophile as a model system to purify stable Dicers for biochemical dissection. While the function of these two Dicers was thought to be partially redundant, our results show that they cleave different RNA substrates, similar to what is observed in the Drosophila melanogaster system. S. thermophile Dicer-1 dices hairpin precursor microRNAs faster than long double-stranded RNAs while S. thermophile Dicer-2 can only cleave perfect RNA duplexes accurately with the requirement for ATP. Removal of the helicase domain from Dicer-2 relieves the requirement of ATP for cleavage and allows the enzyme to process hairpin substrates. In addition, only Dicer-2 shows robust ATP hydrolysis in the presence of dsRNA and partial stimulation in the presence of single-stranded RNA or an RNA/DNA hybrid. The conserved function of these two Dicers may indicate a common mechanism used by eukaryotes to process small RNAs.
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