Selective footprinting of 40S and 80S ribosome subpopulations (Sel-TCP-seq) to study translation and its control

Kód výsledku v IS VaVaI

<a href="https://www.isvavai.cz/riv?ss=detail&h=RIV%2F61388971%3A_____%2F22%3A00562220" target="_blank" >RIV/61388971:_____/22:00562220 - isvavai.cz</a>

Výsledek na webu

<a href="https://www.nature.com/articles/s41596-022-00708-4" target="_blank" >https://www.nature.com/articles/s41596-022-00708-4</a>

DOI - Digital Object Identifier

<a href="http://dx.doi.org/10.1038/s41596-022-00708-4" target="_blank" >10.1038/s41596-022-00708-4</a>

Jazyk výsledku

angličtina

Název v původním jazyce

Selective footprinting of 40S and 80S ribosome subpopulations (Sel-TCP-seq) to study translation and its control

Popis výsledku v původním jazyce

This extension of the TCP-seq protocol describes procedures for selective profiling of 40S and 80S ribosome subpopulations bound by a factor of interest, permitting detailed studies of the different stages of translation in mammalian and yeast cells.nMultiple aspects of mRNA translation are subject to regulation. Here we present a ribosome footprinting protocol to determine the location and composition of 40S and 80S ribosome complexes on endogenous mRNAs transcriptome-wide in vivo in yeast and mammalian cells. We present an extension of the translation complex profiling (TCP-seq) protocol, originally developed in yeast, by including an immunoprecipitation step to assay the location of both 40S and 80S ribosome complexes containing proteins of interest. This yields information on where along mRNAs the ribosome-bound protein of interest joins the ribosome to act, and where it leaves again, thereby monitoring the sequential steps of translation and the roles of various translation factors therein. Rapid fixation of live cells ensures the integrity of all translation complexes bound to mRNA at native positions. Two procedures are described, differing mainly in the fixation conditions and the library preparation. Depending on the research question, either procedure offers advantages. Execution of a Sel-TCP-seq experiment takes 5-10 working days, and initial data analysis can be completed within 2 days.

Název v anglickém jazyce

Selective footprinting of 40S and 80S ribosome subpopulations (Sel-TCP-seq) to study translation and its control

Popis výsledku anglicky

This extension of the TCP-seq protocol describes procedures for selective profiling of 40S and 80S ribosome subpopulations bound by a factor of interest, permitting detailed studies of the different stages of translation in mammalian and yeast cells.nMultiple aspects of mRNA translation are subject to regulation. Here we present a ribosome footprinting protocol to determine the location and composition of 40S and 80S ribosome complexes on endogenous mRNAs transcriptome-wide in vivo in yeast and mammalian cells. We present an extension of the translation complex profiling (TCP-seq) protocol, originally developed in yeast, by including an immunoprecipitation step to assay the location of both 40S and 80S ribosome complexes containing proteins of interest. This yields information on where along mRNAs the ribosome-bound protein of interest joins the ribosome to act, and where it leaves again, thereby monitoring the sequential steps of translation and the roles of various translation factors therein. Rapid fixation of live cells ensures the integrity of all translation complexes bound to mRNA at native positions. Two procedures are described, differing mainly in the fixation conditions and the library preparation. Depending on the research question, either procedure offers advantages. Execution of a Sel-TCP-seq experiment takes 5-10 working days, and initial data analysis can be completed within 2 days.

Druh

J_imp - Článek v periodiku v databázi Web of Science

CEP obor

—

OECD FORD obor

10606 - Microbiology

Projekt

Výsledek vznikl pri realizaci vícero projektů. Více informací v záložce Projekty.

Návaznosti

I - Institucionalni podpora na dlouhodoby koncepcni rozvoj vyzkumne organizace

Rok uplatnění

2022

Kód důvěrnosti údajů

S - Úplné a pravdivé údaje o projektu nepodléhají ochraně podle zvláštních právních předpisů

Název periodika

Nature Protocols

ISSN

1754-2189

e-ISSN

1750-2799

Svazek periodika

17

Číslo periodika v rámci svazku

10

Stát vydavatele periodika

GB - Spojené království Velké Británie a Severního Irska

Počet stran výsledku

49

Strana od-do

2139-2187

Kód UT WoS článku

000828917600001

EID výsledku v databázi Scopus

2-s2.0-85134645475