Journal
ISCIENCE
Volume 25, Issue 7, Pages -Publisher
CELL PRESS
DOI: 10.1016/j.isci.2022.104516
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Funding
- Samuro Kakiuchi Memorial Research Award for Young Scientists
- JSPS [18K14674, 20H03241, 20H04844, 21H05720, 17H05667]
- JST PRESTO [JPMJPR18H2]
- JST FOREST [JPMJFR214L]
- JST ERATO [JPMJER2101]
- Takeda Science Foundation
- AMED (18dm0307023h)
- JST Strategic Basic Research Program CREST [18070870]
- AMED Advanced Research and Development Programs for Medical Innovation CREST [18068699]
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pSNAP is a novel method for proteome-wide profiling of NPCs without the need for ribosome purification and/or chemical labeling, capturing bona fide NPCs and their characteristics. It can be used to evaluate the effect of potential molecular therapies on translation and study modifications on NPCs and their interactions with ribosome-associated factors.
Cellular global translation is often measured using ribosome profiling or quantitative mass spectrometry, but these methods do not provide direct information at the level of elongating nascent polypeptide chains (NPCs) and associated co-translational events. Here, we describe pSNAP, a method for proteome-wide profiling of NPCs by affinity enrichment of puromycin- and stable isotope-labeled polypeptides. pSNAP does not require ribosome purification and/or chemical labeling, and captures bona fide NPCs that characteristically exhibit protein N-terminus-biased positions. We applied pSNAP to evaluate the effect of silmitasertib, a potential molecular therapy for cancer, and revealed acute translational repression through casein kinase II and mTOR pathways. We also characterized modifications on NPCs and demonstrated that the combination of different types of modifications, such as acetylation and phosphorylation in the N-terminal region of histone H1.5, can modulate interactions with ribosome-associated factors. Thus, pSNAP provides a framework for dissecting co-translational regulations on a proteome-wide scale.
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