scholarly journals pSNAP: Proteome-wide analysis of elongating nascent polypeptide chains

2021 ◽  
Author(s):  
Junki Uchiyama ◽  
Rohini Roy ◽  
Dan Ohtan Wang ◽  
Chiaki Yoshino ◽  
Yuichiro Mishima ◽  
...  
Keyword(s):  
Ribosome Profiling ◽  
Molecular Therapy ◽  
Translational Repression ◽  
Quantitative Mass Spectrometry ◽  
Nascent Polypeptide ◽  
Affinity Enrichment ◽  
Different Types ◽  
Wide Scale ◽  
Bona Fide ◽  
Polypeptide Chains

SummaryCellular 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 reaction, 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 COVID-19 patients, 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.

scholarly journals Selective ribosome profiling as a tool for studying the interaction of chaperones and targeting factors with nascent polypeptide chains and ribosomes

2013 ◽  
Vol 8 (11) ◽  
pp. 2212-2239 ◽  
Author(s):  
Annemarie H Becker ◽  
Eugene Oh ◽  
Jonathan S Weissman ◽  
Günter Kramer ◽  
Bernd Bukau
Keyword(s):  
Ribosome Profiling ◽  
Nascent Polypeptide ◽  
Polypeptide Chains

scholarly journals Chromosome segregation fidelity is controlled by small changes in phospho-occupancy at the kinetochore-microtubule interface

2021 ◽  
Author(s):  
Thomas J. Kucharski ◽  
Rufus Hards ◽  
Kristina M. Godek ◽  
Scott A. Gerber ◽  
Duane A. Compton
Keyword(s):  
Error Correction ◽  
Chromosome Segregation ◽  
Dynamic Range ◽  
High Sensitivity ◽  
Cyclin B1 ◽  
Quantitative Mass Spectrometry ◽  
Kinetochore Protein ◽  
Microtubule Attachment ◽  
Different Types ◽  
Narrow Dynamic Range

SummaryKinetochore protein phosphorylation promotes the correction of erroneous microtubule attachments to ensure faithful chromosome segregation during cell division. Determining how phosphorylation executes error correction requires an understanding of whether kinetochore substrates are completely (i.e. all-or-none) or only fractionally phosphorylated. Using quantitative mass spectrometry (MS), we measured phospho-occupancy on the conserved kinetochore protein Hec1 (NDC80) that directly binds microtubules. None of the positions measured exceeded ∼50% phospho-occupancy, and the cumulative phospho-occupancy changed by only ∼20% in response to changes in microtubule attachment status. The narrow dynamic range of phospho-occupancy is maintained by ongoing phosphatase activity. Further, both Cdk1-Cyclin B1 and Aurora kinases phosphorylate Hec1 to enhance error correction in response to different types of microtubule attachment errors. Thus, networks of kinases and phosphatases maintain low inherent phospho-occupancy to promote microtubule attachment to kinetochores while providing for high sensitivity of kinetochore-microtubule attachments to very small changes in phospho-occupancy to ensure high mitotic fidelity.

O-GlcNAc occurs cotranslationally to stabilize nascent polypeptide chains

2015 ◽  
Vol 11 (5) ◽  
pp. 319-325 ◽  
Author(s):  
Yanping Zhu ◽  
Ta-Wei Liu ◽  
Samy Cecioni ◽  
Razieh Eskandari ◽  
Wesley F Zandberg ◽  
...  
Keyword(s):  
Nascent Polypeptide ◽  
Polypeptide Chains

scholarly journals Integrated in vivo and in vitro nascent chain profiling reveals widespread translational pausing

2016 ◽  
Vol 113 (7) ◽  
pp. E829-E838 ◽  
Author(s):  
Yuhei Chadani ◽  
Tatsuya Niwa ◽  
Shinobu Chiba ◽  
Hideki Taguchi ◽  
Koreaki Ito
Keyword(s):  
Escherichia Coli ◽  
Membrane Proteins ◽  
Ribosome Profiling ◽  
Cytosolic Proteins ◽  
Nascent Polypeptide ◽  
Nascent Chain ◽  
Chemical Trait

Although the importance of the nonuniform progression of elongation in translation is well recognized, there have been few attempts to explore this process by directly profiling nascent polypeptides, the relevant intermediates of translation. Such approaches will be essential to complement other approaches, including ribosome profiling, which is extremely powerful but indirect with respect to the actual translation processes. Here, we use the nascent polypeptide's chemical trait of having a covalently attached tRNA moiety to detect translation intermediates. In a case study,Escherichia coliSecA was shown to undergo nascent polypeptide-dependent translational pauses. We then carried out integrated in vivo and in vitro nascent chain profiling (iNP) to characterize 1,038 proteome members ofE.colithat were encoded by the first quarter of the chromosome with respect to their propensities to accumulate polypeptidyl–tRNA intermediates. A majority of them indeed undergo single or multiple pauses, some occurring only in vitro, some occurring only in vivo, and some occurring both in vivo and in vitro. Thus, translational pausing can be intrinsically robust, subject to in vivo alleviation, or require in vivo reinforcement. Cytosolic and membrane proteins tend to experience different classes of pauses; membrane proteins often pause multiple times in vivo. We also note that the solubility of cytosolic proteins correlates with certain categories of pausing. Translational pausing is widespread and diverse in nature.

Folding of nascent polypeptide chains in a high molecular mass assembly with molecular chaperones

Nature ◽  
1994 ◽  
Vol 370 (6485) ◽  
pp. 111-117 ◽  
Author(s):  
Judith Frydman ◽  
Elmar Nimmesgern ◽  
Kenzo Ohtsuka ◽  
F. Ulrich Hartl
Keyword(s):  
Molecular Mass ◽  
Molecular Chaperones ◽  
High Molecular Mass ◽  
Nascent Polypeptide ◽  
Mass Assembly ◽  
Polypeptide Chains
Sign in / Sign up

Export Citation Format

Share Document