Ethan C. Strayer, Srikar Krishna, Haejeong Lee, Charles Vejnar, Nils Neuenkirchen et al.
The cis-regulatory elements encoded in an mRNA determine its stability and translational output. While there has been a considerable effort to understand the factors driving mRNA stability, the regulatory frameworks governing translational control remain more elusive. We have developed a novel massively parallel reporter assay (MPRA) to measure mRNA translation, named Nascent Peptide Translating Ribosome Affinity Purification (NaP-TRAP). NaP-TRAP measures translation in a frame-specific manner through the immunocapture of epitope tagged nascent peptides of reporter mRNAs. We benchmark NaP-TRAP to polysome profiling and use it to quantify Kozak strength and the regulatory landscapes of 5’ UTRs in the developing zebrafish embryo and in human cells. Through this approach we identified general and developmentally dynamic cis-regulatory elements, as well as potential trans-acting proteins. We find that U-rich motifs are general enhancers, and upstream ORFs and GC-rich motifs are global repressors of translation. We also observe a translational switch during the maternal-to-zygotic transition, where C-rich motifs shift from repressors to prominent activators of translation. Conversely, we show that microRNA sites in the 5’ UTR repress translation following the zygotic expression of miR-430. Together these results demonstrate that NaP-TRAP is a versatile, accessible, and powerful method to decode the regulatory functions of UTRs across different systems.
Ethan C. Strayer, Srikar Krishna, Haejeong Lee, Charles Vejnar, Nils Neuenkirchen, Amit Gupta, Jean-Denis Beaudoin, Antonio J. Giraldez. NaP-TRAP reveals the regulatory grammar in 5’UTR-mediated translation regulation during zebrafish development. 2024-12-30. https://doi.org/10.1038/s41467-024-55274-y
A synthetic validation library tested all 256 possible tetramer repeats separated by dinucleotide spacers in the common 5'UTR reporter. In vitro transcribed reporters were injected into single-cell zebrafish embryos and translation was measured at 6 hpf.
A 124-nt library tiled the 5'UTRs of maternally supplied zebrafish transcripts plus IRES controls. The in vitro transcribed library was injected into single-cell embryos and translation was measured at 6 hpf using the SV40 reporter 3' processing configuration.
A 128-reporter synthetic 5'UTR library encoded combinations of no ORFs, uORFs, and overlapping ORFs in three reading frames. The 3xFLAG-HA-MYC reporter was injected into single-cell zebrafish embryos and FLAG, HA, and MYC NaP-TRAP pulldowns were measured at 6 hpf.
The zebrafish 5'UTR tiled reporter library was transfected into HEK293T cells and fractionated by sucrose-gradient polysome profiling 12 hours post-transfection. The table reconstructs the reporter-level fraction distributions and mean ribosome load (MRL) used to benchmark NaP-TRAP.
A 124-nt library tiled the 5'UTRs of maternally supplied zebrafish transcripts plus IRES controls. The in vitro transcribed library was injected into single-cell embryos and translation was measured at 2 hpf using the SV40 reporter 3' processing configuration.
A randomized Kozak library varied six nucleotides upstream and one nucleotide downstream of the AUG in a common 3xFLAG-GFP reporter. In vitro transcribed reporter mRNAs were injected into single-cell Danio rerio embryos and translation was measured by NaP-TRAP at 6 hpf.
A 124-nt library tiled the 5'UTRs of maternally supplied zebrafish transcripts plus IRES controls. The in vitro transcribed library was injected into single-cell embryos and translation was measured at 6 hpf using a reporter with a poly(A) tail.
The zebrafish 5'UTR tiled reporter library was transfected into HEK293T cells and translation was measured 12 hours post-transfection by NaP-TRAP. The table reports reporter-level FLAG pulldown/input translation effects across four biological replicates.
A 124-nt library tiled the 5'UTRs of maternally supplied zebrafish transcripts plus IRES controls. The in vitro transcribed library was injected into single-cell embryos and translation was measured at 2 hpf using a reporter with a poly(A) tail.
A synthetic validation library tested all 256 possible tetramer repeats separated by dinucleotide spacers in the common 5'UTR reporter. In vitro transcribed reporters were injected into single-cell zebrafish embryos and translation was measured at 2 hpf.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 15 files (ZIP)kozak_reporters.famultiframe_reporters.fantrap_kozak_counts.jsonntrap_multiframe_counts.jsonntrap_utr5_fish_pa_sv40_counts.jsonntrap_utr5_hek293t_counts.jsonntrap_validation_counts.jsonpolysome_hek293t_counts.jsonREADME.txtsupplementary_dataset_1.xlsxsupplementary_dataset_2.xlsxsupplementary_dataset_3.xlsxsupplementary_dataset_4.xlsxutr5_fish_reporters.favalidation_reporters.fa