Figure 4. Poly(A) length and ribosome distribution.

(A, B) Single nucleotide resolution electrophoresis for poly(A) length for a target (A) and non-target (B) in wild type and MZdicer (14). A₀ represents the polyadenylation site confirmed by DNA sequencing (fig. S9). (C)GFP expression (green) from an injected miR-430 reporter mRNA containing the 3′UTR for zgc:63829 with wild type (wt-UTR) or mutated (mut-UTR) miR-430 sites. The 3′UTRs are followed by an internal poly(A) tail (A₉₈C₁₀) or a polyadenylation signal. Expression of a co-injected dsRed control mRNA is shown in red. Note the repression of the wild-type reporter compared to the mutant reporter when a internal polyA tail is used. (D) Gel electrophoresis of a PCR to determine the length of the poly(A) tail (ending in A, upper panel) and A₉₈C₁₀ (below). Note the polyadenylation by 2hpf and deadenylation by 6hpf, but deadenylation of the mRNA with the internal polyA tail (A₉₈C₁₀)is delayed. Estimated size of the deadenylated product is shown as (A₀). (E) Two models for translational repression: reducing translation initiation (left) or causing ribosome drop off/slower elongation rate (right). (F) Plot showing relative RPF read density (top) and mRNA density (bottom) along the length of miR-430 targets (red) undergoing >1.5-fold translation repression at 4hpf; and non-targets (gray). Points show mean +/− SEM log2 fold differences between wild type and MZdicer expression in 50nt bins spanning the first and last 1000 nts of the genes (14). Bins represent 41≥N≥277 genes for targets, 107≥N≥906 genes for non targets. Bin values do not significantly differ (p=0.53, Friedman rank sum test).