PUNCH-P for global translatome profiling: Methodology, insights and comparison to other techniques

Translation
Ranen AvinerOrna Elroy-Stein

Abstract

Regulation of mRNA translation is a major modulator of gene expression, allowing cells to fine tune protein levels during growth and differentiation and in response to physiological signals and environmental changes. Mass-spectrometry and RNA-sequencing methods now enable global profiling of the translatome, but these still involve significant analytical and economical limitations. We developed a novel system-wide proteomic approach for direct monitoring of translation, termed PUromycin-associated Nascent CHain Proteomics (PUNCH-P), which is based on the recovery of ribosome-nascent chain complexes from cells or tissues followed by incorporation of biotinylated puromycin into newly-synthesized proteins. Biotinylated proteins are then purified by streptavidin and analyzed by mass-spectrometry. Here we present an overview of PUNCH-P, describe other methodologies for global translatome profiling (pSILAC, BONCAT, TRAP/Ribo-tag, Ribo-seq) and provide conceptual comparisons between these methods. We also show how PUNCH-P data can be combined with mRNA measurements to determine relative translation efficiency for specific mRNAs.

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Citations

Sep 6, 2014·Cell Death and Differentiation·A Wilczynska, M Bushell
Dec 19, 2019·Frontiers in Plant Science·Paula LlabataMarie-Theres Hauser
Dec 4, 2020·Journal of Proteome Research·Linda Berg Luecke, Rebekah L Gundry
Feb 18, 2021·ELife·Luca MinatiMassimiliano Clamer
Mar 30, 2021·Journal of Experimental Botany·Elmien Heyneke, Rainer Hoefgen
Jun 29, 2021·Frontiers in Cell and Developmental Biology·Shivani C KharodYoung J Yoon

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Methods Mentioned

BETA
pull-down
affinity purification
RNA-seq
Ribo-seq

Software Mentioned

PUNCH
Ribo
QuaNCAT
seq
pSILAC
MaxQuant
BONCAT

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