Figure 3. Contribution of single 3′ non-seed nucleotides to the in vivo function of let-7a.

(A) Alignment of pre-miRNA sequences. Boxed 3′ non-seed regions.

(B) Small RNA sequencing reads from the L4 larvae that mapped to WT or mutant let-7a sequences for each single mutant. The reads mapping to WT let-7a for strains carrying mutations at g11–g13 include WT let-7a miRNA from balancer umnIs25(mnDp1). The WT reads from U21A/U22A mutants likely reflect let-7a(mutant) miRNAs whose 3′ ends had been trimmed and subsequently uridylated in vivo, resulting in artificial let-7a(+) reads. Dashed lines, 100% (top) and 50% (bottom) of RPM of let-7a(+) in WT.

(C) Quantitation of let-7a lf phenotypes: percentage of animals with abnormal COL-19:GFP expression (top), numbers of progeny per animal (center), and percentage of adult lethality (bottom). Lethal phenotypes are categorized as in Figure 2L. Abnormal COL-19:GFP patterns are classified as no Hyp7 expression (severe) or faint Hyp7 expression (mild).

(D–G) DIC images of the vulva region in adults at 25°C. Scale bars, 25 μm.

(H) Functional synergy between g18 and the critical non-seed region based on vulva integrity defect. Labels are identical to (C) (bottom).

(I) Summary of the functional merits of let-7a 3′ non-seed nucleotides. Colored circle, 3′ non-seed nucleotide; red proportion, average adult lethality caused by vulva bursting at all temperatures.

Error bars indicate mean ± SD. See also Figures S2 and S3. Details of the phenotypes are available in Table S1.