NaP-TRAP reveals the regulatory grammar in 5'UTR-mediated translation regulation during zebrafish development.

Strayer, Ethan C.; Krishna, Srikar; Lee, Haejeong; Vejnar, Charles; Neuenkirchen, Nils; Gupta, Amit; Beaudoin, Jean-Denis; Giraldez, Antonio J.

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. The cis-regulatory elements encoded in the sequence and structure of mRNAs determine their translational output. Here the authors develop NaP-TRAP, an assay to measure translation in a frame-specific manner, revealing dynamic regulatory elements in zebrafish embryos and HEK293T cells.

HUMAN embryos; PEPTIDES; BRACHYDANIO; MESSENGER RNA; MICRORNA

Nature Communications, 2024, Vol 15, Issue 1, p1

2041-1723

Academic Journal

10.1038/s41467-024-55274-y