References and Notes Materials and Methods Som Text Fig. S1 Table S1 References a Microrna in a Multiple- Turnover Rnai Enzyme Complex

RNA detected 2 and 3 days postinfiltration, respectively, corresponded to the SCL6-IV(b) RNA product. Relatively small proportions of SCL6-IV mRNA were cleaved after coexpression with empty vector (Fig. 2B, top, lanes 4 and 5), IGR-mi39 (lanes 6 and 7), and 35S:AS-IGR-mi39 constructs (lanes 10 and 11), which can be explained by the basal or low levels of miRNA 39 in these samples. No cleavage products were detected with the SCL6-IV 5Ј-end probe (Fig. 2B), suggesting that the 5Ј fragment was less stable than the 3Ј fragment [SCL6-IV(b)]. Identical results were obtained when the SCL6-III mRNA was used as a target in coexpression assays (23). To determine whether cleavage of SCL6-IV mRNA depends on perfect complementa-rity with miRNA 39, we introduced three mismatches into the sequence complementary to miRNA 39 in SCL6-IV RNA (construct 35S:SCL6-IV/mut39) (Fig. 3). Whereas the wild-type SCL6-IV mRNA was cleaved efficiently in the presence of the miRNA 39 – producing 35S:IGR-mi39 construct (Fig. 3, lanes 8, 9, 18, and 19), the SCL6-IV/mut39 mRNA was completely resistant to cleavage (lanes 10, 11, 20, and 21). Furthermore, the low level of cleavage of SCL6-IV mRNA in the presence of the empty vector construct was inhibited by the mutations (Fig. 3, lanes 4 to 7), confirming that this activity was due to low levels of endogenous miRNA 39 in N. benthamiana tissue. The finding of miRNA-directed cleavage of several SCL mRNA targets in Arabidopsis indicates that there are at least two functional classes of miRNAs. Members of the small temporal RNA (stRNA)–like class, including C. elegans lin-4 and let-7 miRNAs, down-regulate translation of target mRNAs but do not direct RNA target degradation or site-specific cleavage (7, 8, 10, 11). The stRNA class members do not interact with perfect complementarity to their natural targets, which may explain why they do not exhibit siRNA-like activity (24). In contrast, members of a class represented by Arabidopsis miRNA 39 interact with perfect complemen-tarity and appear to mimic siRNA function to guide cleavage. We propose that miRNA 39 incorporates into a RISC-like complex identical or similar to the RISC complex that mediates target cleavage during RNA silencing (21, 22). Support for this concept also comes from the finding that engineered miRNA-target combinations with perfect complementarity result in target RNA cleav-age (25, 26). Finally, miRNA 39 –guided cleavage of mRNAs has several possible consequences , including developmentally coordinated inactivation of SCL mRNAs. Internal cleavage might also …

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