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<p>Producing functional proteins involves multiple steps during mRNA translation on the ribosomes. However, co-translational regulatory mechanisms remain poorly characterized in intact mammalian systems. As a proof-of-concept, we developed a multi-omics approach to investigate endothelial-specific, co-translational regulation by modifying the translating ribosome affinity purification (TRAP) in vivo. We simultaneously co-immunoprecipitated (IP) polysome-associated mRNAs and proteins from the hearts of hemagglutinin-tagged ribosomal protein L22 mice (RiboTag) crossed with inducible endothelial-specific <i>Cdh5CreERT2</i> mice (<i>RiboTag</i><sub><i>EC</i></sub>). To perturb endothelial function, female mice were injected with E. <i>coli</i> lipopolysaccharide (LPS) (6&#xa0;mg/Kg, i.p., 12&#xa0;h). Hearts were homogenized, with ~ 10% used for input RNA-Seq and proteomics controls, and the remainder for IP of ribosome-bound polyadenylated mRNA and proteins. Endothelial cell transcripts (<i>pecam1</i>, <i>cdh5</i>) were enriched &gt; 5-fold, while markers characteristic of other cell types were significantly depleted (&lt; 0.05 q-value). We aligned transcriptomic and proteomic datasets (&gt; 1250 overlapping terms) to identify pathways associated with concordant and discordant co-translational regulation. LPS was identified as the upstream regulator of the co-translational dataset that was concordantly regulated. Upregulated mRNAs but not proteins related to glycolysis were discordantly regulated. These findings validate our proof-of-concept multi-omics approach as a predictive platform for identifying disease-relevant pathways regulated at the co-translational level <i>in vivo.</i></p>

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Co-translational profiling in the cardiac endothelium in response to LPS-induced inflammation in female mice in vivo: a proof-of-concept approach

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年4月28日

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  • Chad M. Warren,
  • Bhairavi Swaminathan,
  • Paulina Langa,
  • Stephanie R. Villa,
  • Walter C. Thompson,
  • Magdalena Chrzanowska,
  • Jan K. Kitajewski,
  • R. John Solaro,
  • Beata M. Wolska,
  • Paul H. Goldspink

摘要

Producing functional proteins involves multiple steps during mRNA translation on the ribosomes. However, co-translational regulatory mechanisms remain poorly characterized in intact mammalian systems. As a proof-of-concept, we developed a multi-omics approach to investigate endothelial-specific, co-translational regulation by modifying the translating ribosome affinity purification (TRAP) in vivo. We simultaneously co-immunoprecipitated (IP) polysome-associated mRNAs and proteins from the hearts of hemagglutinin-tagged ribosomal protein L22 mice (RiboTag) crossed with inducible endothelial-specific Cdh5CreERT2 mice (RiboTagEC). To perturb endothelial function, female mice were injected with E. coli lipopolysaccharide (LPS) (6 mg/Kg, i.p., 12 h). Hearts were homogenized, with ~ 10% used for input RNA-Seq and proteomics controls, and the remainder for IP of ribosome-bound polyadenylated mRNA and proteins. Endothelial cell transcripts (pecam1, cdh5) were enriched > 5-fold, while markers characteristic of other cell types were significantly depleted (< 0.05 q-value). We aligned transcriptomic and proteomic datasets (> 1250 overlapping terms) to identify pathways associated with concordant and discordant co-translational regulation. LPS was identified as the upstream regulator of the co-translational dataset that was concordantly regulated. Upregulated mRNAs but not proteins related to glycolysis were discordantly regulated. These findings validate our proof-of-concept multi-omics approach as a predictive platform for identifying disease-relevant pathways regulated at the co-translational level in vivo.