dsRNA templates were cleaved in vitro with dicer-like enzymes and the products were sequenced

We characterize three Dicer-like enzymes involved in Paramecium development, two of which show sequence cleavage preferences for their RNA templates. Dicer proteins are known to produce small RNAs such as miRNAs and siRNAs from long dsRNA templates, which they cleave into specific and often precise lengths. sRNAs generated by Dicer proteins are bound by AGO proteins, which have the capability to select the guide strand from passenger strand and which sometimes also show 5’ end preferences. However, Dicer proteins themselves have never been shown to have sequence preferences. In Paramecium development, two classes of small RNAs are produced by three Dicer-like enzymes, Dcl2, Dcl3 and Dcl5. Dcl2 and 3 cooperate to produce 25 nt long scan RNAs with a 5’ UNG signature, while Dcl5 produces RNAs of varying lengths with 5’ UAG and 3’ CNAU signatures. Through in vitro assays, we demonstrate that Dcl2 has a strict size preference for 25 nt, while Dcl3 has a sequence preference for 5’ UNG. They are thus both needed to produce mature scan RNAs. Dcl5, meanwhile, has cleavage preferences for both its 5’ and 3’ ends, which leads to the recognition of RNAs corresponding to excised DNA elements as discrete units. The identification of enzymes with the capability of cleaving RNA at specific sequences within a long dsRNA template provides an insight into the diversity of mechanisms involved in RNA processing and offers the opportunity for future protein engineering to produce enzymes with any RNA sequence preference.