Figure 6. Molecular determinants of miRNA turnover.

(A) In Drosophila, miRNAs preferentially load into Ago1 to form miRISC. At the same time, a subset of miR*s load onto Ago2 to form siRISC, prompting the methylation of Ago2-bound small RNAs at the 2´ position of the 3´-terminal ribose. If and how Argonaute protein identity influences small RNA stability remains unknown. (B) Median decay kinetics of Ago2-enriched miRNAs (n=8; classified in Figure S6B) in an 4sU metabolic labeling time course in wild-type (wt, black) or ago2^ko S2 cells (red) or from wild-type S2 cells employing a cloning strategy that enriches for small RNAs with modified 3´ termini (wt oxidized, blue). Median and interquartile range of two-phase or one-phase saturation kinetics (as specified in main text) are shown. The half-lives (t_½) as determined by curve-fitting are indicated. (C) Comparison of small RNA stabilities in the context of Ago1 and Ago2. Half-lives of the 30 most abundant Ago1-bound miRNAs (red, Ago1) compared to the most abundant miRs and miR*s in small RNA libraries employing a cloning strategy that enriches for small RNAs with modified 3´ termini (blue, Ago2). The median and interquartile range is indicated. P-value (Mann-Whitney test) is shown. (D) Half-lives of miRNAs subjected to exonucleolytic 3´ end trimming by Nbr (in red, n=25) and non-Nbr substrates (in black, n=17) as classified in Figure S4. Median and interquartile range are shown. P-value (Mann-Whitney test) is indicated. See also Figure S6.