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Updated: Sep 6, 2025
10:15
Isolation of Ribosome Bound Nascent Polypeptides in vitro to Identify Translational Pause Sites Along mRNA
Published on: July 6, 2012
16.4K
pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains.
Junki Uchiyama1, Rohini Roy2,3,4, Dan Ohtan Wang2,3,4,5
1Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto 606-8501, Japan.
Iscience
|June 27, 2022
Summary
We developed pSNAP, a new method to study nascent polypeptide chains (NPCs) during protein synthesis. This technique reveals how cancer drugs affect translation and identifies protein modifications impacting ribosome interactions.
Area of Science:
- Molecular Biology
- Proteomics
- Biochemistry
Background:
- Existing methods like ribosome profiling and mass spectrometry lack direct insight into elongating nascent polypeptide chains (NPCs) and co-translational events.
- Understanding NPC dynamics is crucial for deciphering protein synthesis regulation.
Purpose of the Study:
- To introduce pSNAP, a novel method for proteome-wide profiling of NPCs.
- To analyze co-translational events and protein modifications with high precision.
- To investigate the impact of cancer therapeutics on cellular translation.
Main Methods:
- pSNAP utilizes affinity enrichment of puromycin- and stable isotope-labeled polypeptides to profile NPCs.
- The method does not require ribosome purification or chemical labeling.
- It captures bona fide NPCs, characterized by N-terminus-biased positions.
Main Results:
- pSNAP successfully profiled NPCs, revealing acute translational repression by silmitasertib via casein kinase II and mTOR pathways.
- Characterization of NPC modifications, including acetylation and phosphorylation on histone H1.5, demonstrated their role in modulating ribosome-associated factor interactions.
Conclusions:
- pSNAP offers a powerful framework for dissecting co-translational regulations at the proteome-wide level.
- The method provides new insights into cancer therapy mechanisms and post-translational modifications during translation.
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