Pratibha Jagannatha1,2,3,4, Alexandra T. Tankka1,2,3, Daniel A. Lorenz5, Tao Yu1,2,3, Brian A. Yee1,2,3, Kristopher W. Brannan1,2,3,7, Cathy J. Zhou1,2,3, Jason G. Underwood6 and Gene W. Yeo1,2,3,4,5 1Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA; 2Sanford Stem Cell Institution Innovation Center and Stem Cell Program, University of California San Diego, La Jolla, California 92037, USA; 3Institute for Genomic Medicine, University of California San Diego, La Jolla, California 92093, USA; 4Bioinformatics and Systems Biology Graduate Program, University of California San Diego, La Jolla, California 92093, USA; 5Sanford Laboratories for Innovative Medicine, La Jolla, California 92121, USA; 6PacBio, Menlo Park, California 94025, USA

↵7 Present address: Center for RNA Therapeutics, Department of Cardiovascular Sciences, Houston Methodist Research Institute, Houston, TX 77030, USA

Corresponding author: geneyeoucsd.edu Abstract

Transcription and translation are intertwined processes in which mRNA isoforms are crucial intermediaries. However, methodological limitations in analyzing translation at the mRNA isoform level have left gaps in our understanding of critical biological processes. To address these gaps, we developed an integrated computational and experimental framework called long-read Ribo-STAMP (LR-Ribo-STAMP) that capitalizes on advancements in long-read sequencing and RNA-base editing–mediated technologies to simultaneously profile translation and transcription at both the gene and mRNA isoform levels. We also developed the EditsC metric to quantify editing and leverage the single-molecule, full-length transcript information provided by long-read sequencing. Here, we report concordance between gene-level translation profiles obtained with long-read and short-read Ribo-STAMP. We show that LR-Ribo-STAMP successfully profiles translation of mRNA isoforms and links regulatory features, such as upstream open reading frames (uORFs), to translation measurements. We apply LR-Ribo-STAMP to discovering translational differences at both the gene and isoform levels in a triple-negative breast cancer cell line under normoxia and hypoxia and find that LR-Ribo-STAMP effectively delineates orthogonal transcriptional and translation shifts between conditions. We also discover regulatory elements that distinguish translational differences at the isoform level. We highlight GRK6, in which hypoxia is observed to increase expression and translation of a shorter mRNA isoform, giving rise to a truncated protein without the AGC Kinase domain. Overall, LR-Ribo-STAMP is an important advance in our repertoire of methods that measures mRNA translation with isoform sensitivity.

Footnotes

[Supplemental material is available for this article.]

Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.279176.124.

Freely available online through the Genome Research Open Access option.

Received February 22, 2024. Accepted May 31, 2024.