NaP-TRAP: A Versatile and Accessible Workflow to Dissect Principles of Translational Regulation and mRNA Stability.

The translation of messenger RNA (mRNA) into protein is tightly regulated by both cellular trans-factors and cis-regulatory elements encoded within transcripts. Although transcript fate can be measured by transcript abundance or translation efficiency, separating the contribution of individual cis-element within a single transcript is an ongoing challenge. Current, massively parallel reporter assay (MPRA) approaches enable systematic interrogation of cis-regulatory elements that control transcript stability, but translation-focused MPRAs remain technically limited and often inaccessible. Here we present Nascent Peptide Translating Ribosome Affinity Purification (NaP-TRAP), a reporter-based approach that simultaneously measures translation and mRNA abundance. Unlike previous methods, NaP-TRAP captures translation via immunoprecipitation of epitope-tagged nascent peptide chains, providing instantaneous, frame-specific readouts without specialized instrumentation. The method is scalable from single reporters to complex libraries, and adaptable across in vivo and in vitro systems. NaP-TRAP is versatile, allowing assessment of cis-regulatory impact of elements distributed throughout the mRNA, from cap-to-tail. This article covers experimental design, reporter construction, sample processing, and computational analysis for both low- and high-throughput applications. Benchwork can be completed in 4 to 5 days, with quantitative polymerase chain reaction (qPCR)-based readouts requiring only basic Microsoft Excel skills for data processing. Sequencing-based readouts require command-line and Python skills and add 2 to 3 days. NaP-TRAP thus offers an accessible, robust, and quantitative platform to decode the regulatory logic of mRNA translation and stability in diverse biological contexts. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Design, assembly, and synthesis of NaP-TRAP reporter libraries Support Protocol: Design, assembly, and synthesis of NaP-TRAP individual reporters and spike-ins Basic Protocol 2: NaP-TRAP delivery by micro-injection in zebrafish embryos Alternate Protocol 1: NaP-TRAP delivery by transfection in cultured mammalian cells Basic Protocol 3: NaP-TRAP pulldown and RNA extraction Basic Protocol 4: Preparation of NaP-TRAP sequencing libraries Alternate Protocol 2: NaP-TRAP qPCR module for low-cost validation Basic Protocol 5: Computational analysis of NaP-TRAP MPRA data.