Our results show that budding-yeast Dicers produce siRNAs through a mechanism different from that of canonical Dicers. Instead of successively removing siRNA duplexes from the dsRNA termini, Dcr1 starts in the interior and works outward (). This inside-out mechanism initiates with a dimer binding at an arbitrary position within the dsRNA, followed by the recruitment of additional dimers to adjacent sites. As binding propagates in both directions along the dsRNA, slow product release prevents cleavage events from disrupting maintenance of the phase. Cleavage by a collection of aligned dimers precisely generates 23 nt siRNA products paired to each other with 2 nt 3' overhangs.
Inside-Out Mechanism of Budding-Yeast Dicers
The mechanism for Dcr1-catalyzed siRNA production represents a natural example of a molecular ruler that is defined by the spacing of adjacent active sites. The concept of such a molecular ruler has been proposed but then rejected for other enzymes. The multimeric proteasome was hypothesized to generate short peptide products with a length determined by the distance between active sites (Wenzel et al., 1994
), but subsequent experiments ruled out this model (Nussbaum et al., 1998
). Ironically, models for product length determination based on active-site spacing were also proposed for both bacterial RNase III and canonical Dicers (Blaszczyk et al., 2001
; Carmell and Hannon, 2004
), but further study of these enzymes proved these models to be incorrect (Zhang et al., 2004
). Nonetheless, under certain conditions E. coli
RNase III can process long dsRNA into ~23 nt products in vitro
by using a mechanism that might resemble the inside-out mechanism described here (Xiao et al., 2009
). Products of this in vitro
reaction act as potent siRNAs for mammalian gene knock-down (Yang et al., 2002
; Xiao et al., 2009
), as do siRNAs generated by budding-yeast Dicer (Figure S7
In canonical siRNA-generating Dicers, the helicase domain uses ATP to facilitate complete processing of a duplex into siRNAs before beginning on the next duplex (Cenik et al., 2011
; Welker et al., 2011
). In budding-yeast Dicers, cooperativity could facilitate complete processing without requiring such a domain. dsRBD1 and VL-1/2 are candidates for forming cooperative interactions between adjacent Dcr1 dimers bound to dsRNA. Given their roles in dsRNA binding (Figure S1D
), dissecting their potential contributions to cooperativity awaits a high-resolution view of the Dcr1–dsRNA complex, which would reveal dimer–dimer interactions that might be abolished without perturbing dimer–dsRNA interactions. In addition to mediating cooperativity, dimer–dimer interactions might allosterically activate adjacent dimers for cleavage, which would further favor productive cleavage.
In the current RNase III enzyme classification, which is based on domain architecture, class I includes both bacterial RNase III and yeast Rnt1 (MacRae and Doudna, 2007
). We found that despite having similar domain architectures, bacterial RNase III and yeast Rnt1/Dcr1 use distinct active-site arrangements comprising four and six residues, respectively (). Adding this feature to the existing classification criteria would divide RNase III enzymes into four classes more parsimonious with their evolutionary relationships: bacterial RNase III, class I; Drosha, class II; canonical Dicer, class IIIa; and yeast RNase III, including both Rnt1 and Dcr1, class IIIb (). Despite its closer evolutionary relationship to Dcr1, Rnt1 behaves as a molecular ruler in a manner more analogous to canonical Dicer. Just as the canonical Dicer PAZ domain binds to the 2 nt 3' overhang of its substrate to position the RNase III active sites at a defined distance, the Rnt1 dsRBD recognizes the AGNN tetraloop of its substrate to position its active sites for precise cleavage (MacRae and Doudna, 2007
). Thus, the terminus-independent measuring mechanism of Dcr1 departs from the principles operating in other class III RNase III enzymes.
The distinct mechanisms employed by canonical and budding-yeast Dicers to generate similarly sized siRNAs provide a striking example of convergent functional evolution. Although both mechanisms produce siRNAs, the canonical mechanism is more suitable for producing small RNAs that must be processed in a defined register, such as microRNAs and trans-acting siRNAs (Vaucheret, 2005
). In contrast, the inside-out mechanism is more suitable for substrates that lack free helical ends, such as covalently closed molecules (e.g., viroids), dsRNA intermediates of rolling-circle replication, dsRNA with protected termini (e.g., viral ribonucleoproteins), and dsRNA with long single-stranded extensions, including endogenous Dcr1 substrates (Figure S5D
). Thus, in a budding-yeast ancestor, the presence of dsRNA species resistant to processing by the canonical Dicer might have favored the evolution of an additional RNase III enzyme able to pre-process these substrates by cutting in their interior, thereby producing suitable substrates for canonical Dicer. After this enzyme acquired features that enabled it to produce siRNAs on its own, the absence of a phased small-RNA pathway might have allowed loss of the canonical Dicer without deleterious effects, thereby explaining the replacement of the canonical Dicer in the budding-yeast clade.