let-7 rescue is retained by central seed mutations while non-seed mutant effects support potential noncanonical contributions to let-7 function. (A) Partial let-7 precursor structure with the mature let-7 microRNA highlighted (yellow box). Red and blue lines represent Watson–Crick base pairs (C:G and A:U, respectively), with green lines representing wobble base pairs (G:U). (B) Percentages of transgenic animals exhibiting rescue of bursting phenotype, egg-laying phenotype, and adult alae formation for each let7_m construct. “n” refers to total number of transgenic animals assayed for each let7_m construct, with the subsequent value in parentheses referring to the total number of independent transgenic lines assayed. (*) High-frequency bursting of transgenic let7_seedmut animals precludes the assay for adult alae formation in young adults. (C) Structures of two complex let-7 variants. (i) The let7_seedmut construct consists of a multiple seed mutation with five substitutions (shown in red letters) within the seven base seed; black letters are unchanged from WT let-7 sequence. The seed sequence is highlighted by the light blue box. (ii) The let7_strandswap mutation swaps the let-7 microRNA sequence from the 5′ to the 3′ position in the precursor. Yellow boxes highlight the expected small RNA produced from either arm ([top] wild-type let-7*, [bottom] wild-type let-7). The lowercase letters are the unchanged WT let-7 sequence (D) Rescue activities of let7_m constructs. The seed sequence is highlighted by the light blue box. The different colored boxes below let-7 sequence represent different levels of let-7 rescue ([gray] 0.0%, [yellow] 50.0%, [green] 100%) corresponding to the let7_m mutation depicted in the box at the same position. (i) Rescue of vulval bursting phenotype. (ii) Rescue of egg-laying and adult alae formation phenotypes. The top row of colored boxes refers to rescue of egg-laying while the bottom row refers to rescue of adult alae formation in young adults. The numbers within the colored boxes are the average percentages of transgenic mutant animals showing specific rescue activity for let7_m constructs. (E) Binding sites in lin-41 3′ UTR (bottom) are aligned against let-7 mature microRNA (top) (Reinhart et al. 2000; Lewis et al. 2005; Bartel 2009; Jan et al. 2011; Ecsedi et al. 2015). Dots indicate absent bases at those positions; dashes indicate one or more noncomplementary bases. Lowercase letters represent complementary bases as a result of mutations made in let7_m constructs. First miRNA nucleotides and alignments of 5′ ends of binding sites have been included for completeness although their functionalities have not been demonstrated. Colored bars represent levels of let-7 activities ([gray] 0.0%, [yellow] 50.0%, [green] 100%; [top set] rescue of egg-laying, [bottom set] rescue of adult alae formation) for mutations at specific positions. The multiple mutations are not depicted here.
