Flow-based In Vivo Method to Enumerate Translating Ribosomes and Translation Elongation Rate

Area of Science:

• Molecular Biology
• Cellular Biology
• Biochemistry

Background:

• Protein synthesis is a critical, energy-intensive process in rapidly dividing cells.
• Existing methods for quantifying translating ribosomes and their mRNA transit rates are technically demanding, expensive, and provide limited information on translation status.
• Accurate measurement of ribosome activity is crucial for understanding cellular function and disease.

Purpose of the Study:

• To develop and validate a novel, streamlined protocol for quantifying translating ribosome numbers and mRNA transit rates in vivo.
• To enable the detection of ribosome stalling within single cells using flow cytometry.
• To provide a more accessible and efficient method for studying protein synthesis dynamics.

Main Methods:

• Development of a protocol involving intravenous administration of puromycylation, harringtonine, and cycloheximide to mice.
• Ex vivo cell fixation and permeabilization followed by flow cytometry analysis.
• Utilizing a puromycin-specific antibody for quantifying translating ribosome numbers and transit rates.

Main Results:

• The developed protocol allows for the relative quantification of translating ribosomes in mixed single-cell preparations.
• The method enables the relative quantitation of in vivo ribosome transit rates in mixed single-cell preparations.
• The protocol is capable of detecting ribosome stalling in vivo.

Conclusions:

• This novel method offers a robust and accessible approach to measure translating ribosome numbers and mRNA transit rates in vivo.
• The technique facilitates the study of protein synthesis dynamics and ribosome stalling in various biological contexts.
• This advancement provides a valuable tool for researchers investigating cellular energetics and rapidly dividing cells.