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Related Concept Videos

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Long-read Ribo-STAMP simultaneously measures transcription and translation with isoform resolution.

Pratibha Jagannatha1,2,3,4, Alexandra T Tankka1,2,3, Daniel A Lorenz5

  • 1Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, California 92093, USA.

Genome Research
|June 21, 2024
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Summary

We developed long-read Ribo-STAMP (LR-Ribo-STAMP) to profile mRNA translation at the isoform level. This method reveals how gene expression and translation shift in triple-negative breast cancer cells under different oxygen conditions.

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Area of Science:

  • Molecular Biology
  • Genomics
  • Cancer Research

Background:

  • Transcription and translation are fundamental biological processes, with mRNA isoforms acting as key intermediates.
  • Current methods face limitations in analyzing translation at the mRNA isoform level, hindering a comprehensive understanding of gene regulation.
  • Existing techniques often fail to capture the full spectrum of translational control across different mRNA variants.

Purpose of the Study:

  • To develop an integrated computational and experimental framework for simultaneous profiling of transcription and translation at both gene and mRNA isoform levels.
  • To introduce a novel metric, EditsC, for quantifying RNA editing using single-molecule, full-length transcript data.
  • To investigate translational regulation and its link to regulatory features like upstream open reading frames (uORFs).

Main Methods:

  • Development of long-read Ribo-STAMP (LR-Ribo-STAMP), combining long-read sequencing with RNA-base editing.
  • Simultaneous profiling of transcription and translation at gene and mRNA isoform levels.
  • Application of the EditsC metric to quantify RNA editing events.

Main Results:

  • LR-Ribo-STAMP demonstrates concordance with existing short-read methods at the gene level.
  • The framework successfully profiles mRNA isoform translation and links regulatory elements like uORFs to translation.
  • Analysis of triple-negative breast cancer cells under normoxia and hypoxia revealed distinct transcriptional and translational shifts, with LR-Ribo-STAMP effectively delineating these changes.
  • Discovery of specific regulatory elements distinguishing isoform-level translational differences, exemplified by GRK6 where hypoxia promotes a truncated protein isoform.

Conclusions:

  • LR-Ribo-STAMP provides a powerful new method for measuring mRNA translation with high isoform sensitivity.
  • The framework enables the discovery of novel regulatory mechanisms at both the gene and mRNA isoform levels.
  • This advancement offers deeper insights into gene regulation in complex biological processes and diseases like cancer.