Figure 2. In vivo zebrafish 5ÚTR MPRA uncovers translation initiation regulation.

(A) Schematics of the 5´ UTR massively parallel reporter assay (MPRA) employed. The pooled mRNA reporter library was injected into 1-cell stage embryos and 100 embryos were collected at 2, 4, 6 and 10 hours post-fertilization (hpf). Reporter transcripts were separated based on the number of ribosomes bound by sucrose density gradient centrifugation followed by polysome fractionation. Translational behaviours for each reporter 5´ UTR sequence were determined by high-throughput sequencing. (B) Polysome profiling traces for one of the three replicate experiments. The 80S, low molecular weight (LMW) and high molecular weight (HMW) fractions were collected at 2, 4, 6 and 10 hpf and (C) total RNA was extracted for each of the fractions as well as input (total) sample for quantification of relative 5´ UTR reporter abundance (TPM, transcript per million). Ribosome recruitment scores (RRSs) were calculated for each fraction at each developmental time-point. (D) Heatmap representing mean log₂ transformed RRS values of three replicate experiments (n = 17,879). Nine clusters were generated using hierarchical clustering by 5´ UTR sequence (rows) and ordered by fraction and developmental time-point (columns). (E) Density plots of mean log₂ transformed RRS values for three representative clusters displaying distinct ribosome recruitment dynamics. (F) Bar plots of mean log₂ transformed RRS values for a repressive (egfl6) and an enhancing (hnrnpl) 5´ UTR. Error bars represent SD of three replicate experiments. (G) Fluorescence microscopy images of representative embryos at 3 and 8 hpf that were injected with either the egfl6–5ÚTR-sfGFP or the hnrnpl-5ÚTR-sfGFP reporter and a control mCherry reporter at the 1-cell stage. TL – transmitted light. (H) Swarm plot displaying relative fluorescence intensities of 3 and 8 hpf embryos injected with each reporter at the 1-cell stage. sfGFP fluorescence was normalized to mCherry intensity in each embryo. Red bars represent median value, each dot represents one embryo (n = 40) from 2 independent experiments (N = 2), **** p-value < 0.0001 Wilcoxon matched-pairs signed rank test. See also Figure S2, Table S2 and STAR Methods.