Journal: bioRxiv

Article Title: NaP-TRAP: A versatile and accessible workflow to dissect principles of translational regulation and mRNA stability

doi: 10.64898/2026.04.12.718002

Figure Lengend Snippet: Overview of the NaP-TRAP workflow and examples of use cases. ( A ) NaP-TRAP can be performed using either complex reporter libraries or individual reporters, delivered in vivo by zebrafish embryo microinjection or in vitro by mammalian cell transfection, followed by pulldown, RNA purification, and either sequencing- or qPCR-based readout. ( B ) Principle of NaP-TRAP: FLAG-tagged nascent peptides on actively translating ribosomes are immunocaptured to enrich ribosome-associated reporter mRNAs (Pulldown). Reporter abundance is measured in the Input, and translation output is quantified as Pulldown/Input (NaP-TRAP TE). ( C ) Robust delivery and readout across a 100-fold range of injected reporter amounts in zebrafish embryos, with consistent NaP-TRAP TE across all doses. ( D ) Comparable reporter trends across mammalian cell types illustrated by differential translation of control versus oORF-containing reporters in HEK293T, H9, and MCF7 cells. ( E ) NaP-TRAP supports interrogation of diverse regulatory features across the reporter mRNA, including mRNA cap types, 5′-UTR elements (e.g., uORFs/oORFs), coding sequence codon optimality, 3′-UTR elements (e.g., miR-430 sites), and poly(A) tail length in zebrafish embryos. Cartoon diagrams were created individually in BioRender (Smith, J. (2025). BioRender.com/c248457 ).

Article Snippet: Cultured cell lines We have used NaP-TRAP on HEK293T (RRID: CVCL_0063, ATCC Cat. No. CRL-3216) but successfully performed it in various other cell lines including MCF7 (RRID: CVCL_0031, ATCC Cat. No. HTB-22) and H9 (RRID:CVCL_1240, ATCC Cat. No. HTB-176) .

Techniques: In Vivo, Microinjection, In Vitro, Transfection, Purification, Sequencing, Injection, Control

Journal: bioRxiv

Article Title: NaP-TRAP: A versatile and accessible workflow to dissect principles of translational regulation and mRNA stability

doi: 10.64898/2026.04.12.718002

Figure Lengend Snippet: Expected results from NaP-TRAP MPRA analysis pipeline. ( A ) Example histogram of per-insert read counts for one Input replicate with 7,839,828 reads. ( B ) Representative replicate-to-replicate correlations of NaP-TRAP translation efficiency (TE; Pulldown/Input) at 2 hpf and 6 hpf ( R values are Pearson correlations). ( C ) Hierarchical clustering heatmap of Pearson correlations showing TE similarity across replicates and conditions (zebrafish 2 hpf, zebrafish 6 hpf, and HEK293T). ( D ) Distribution of NaP-TRAP TE values at 2 hpf, with the top and bottom 10% of reporters highlighted as activated (blue) and repressed (orange), respectively. ( E ) Example k-mer enrichment analysis for activated and repressed reporter sets at 2 hpf. ( F ) Scatter plot comparing reporter NaP-TRAP TE at 2 versus 6 hpf, highlighting four reporter sets: reporters activated (blue) and repressed (orange) at both stages, reporters with higher TE at 2 hpf than at 6 hpf (2 hpf activated; green), and reporters with high TE at 6 hpf than at 2 hpf (6 hpf activated; pink). ( G ) k-mer enrichment analyses for each of the four reporter sets defined in (F): 2 hpf activated (green), 6 hpf activated (pink), globally repressed (orange) and globally activated (blue).

Article Snippet: Cultured cell lines We have used NaP-TRAP on HEK293T (RRID: CVCL_0063, ATCC Cat. No. CRL-3216) but successfully performed it in various other cell lines including MCF7 (RRID: CVCL_0031, ATCC Cat. No. HTB-22) and H9 (RRID:CVCL_1240, ATCC Cat. No. HTB-176) .

Techniques: