Figure 3.

Imperfect seed matches and extensive 3′ pairing confer target specificity. (A–C) Reporter quantification as in Figure 2, from which the negative control data (black dots) are also replotted for reference; gray shading is bounded by the min-max values of the negative control. (A) The ‘unc-54 + let-7 sites 13–16miR-48-ized’ reporter contains let-7 complementary sites modified to pair miR-48 at position 13–16 but not other seed-distal nucleotides (gray dots). Results from the unmodified ‘unc-54 + let-7 sites’ reporter in wild-type and let-7ts mutant background are from Figure 2C and included for reference (cyan dots). (B) The ‘unc-54 + miR-48 sites’ reporter combines extensive seed-distal complementarity to miR-48 with seed match imperfections whereas (C) the ‘unc-54 + miR-48 sites_perfect seed match’ reporter contains extensive seed-distal complementarity to miR-48 and perfect seed matches. Horizontal line and error bars indicate mean values per condition ± SD. *P < 0.05 and ***P < 0.001, two-tailed unpaired t-test. (D) Animals carrying the lin-41(ap427[dot-1.1_G: U] (9)) allele die in the absence of miR-48. (E) Survival of strain lin-41(xe76[dot-1.1_W-C] upon manipulation of let-7 and miR-48 activity. In this strain, a U at position 8 in the two target sites of the lin-41(ap427[dot-1.1_G:U]) allele has been converted to a C, to permit Watson-Crick instead of G:U wobble base-pairing with the let-7 family seed sequence (Supplementary Figure S2C and D). This allele was crossed into a (i) let-7ts, (ii) mir-48(–) or (iii) mir-48(–) let-7(++) background, where let-7(++) denotes let-7 overexpression from a single copy integrated transgene. Insets magnify the central part of the animal body to reveal egg retention (arrow), i.e. and egg-laying defective (Egl) phenotype. let-7ts: let-7(n2853) X, temperature-sensitive lesion, grown at the restrictive temperature 25°C; mir-48(–): mir-48(n4097) V; mir-48/241/84(–): mir-48/mir-241(ndf51) V, mir-84(n4037) X.