NaP-TRAP, a novel massively parallel reporter assay to quantify translation control

Abstract

Thecis-regulatory elements encoded in a 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 elusive. We have developed a novel massively parallel reporter assay (MPRA) to measure mRNA translation, 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. In contrast to existing MPRA methods, NaP-TRAP does not require specialized equipment and is readily adaptable to steady-state and dynamic model systems. We have employed NaP-TRAP to quantify Kozak strength and the regulatory landscapes of 5’ UTRs in the developing zebrafish embryo and in human cells, characterizing general and developmentally dynamiccis-regulatory elements. To this end, we identify U-rich motifs as general enhancers, and upstream ORFs and GC-rich motifs as 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.