RAFTER: a releasing factor tethered RNA editing system for quantifying mRNA translation

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Current methods for measuring mRNA translation, like polysome profiling, may not accurately reflect translation efficiency.
• Quantifying polysome-associated RNAs doesn't always correlate with the actual rate of protein synthesis.

Purpose of the Study:

• To develop a novel method for precisely measuring mRNA translation levels.
• To introduce a technique that specifically labels successfully translated RNAs.
• To enable high-throughput and single-cell level translation analysis.

Main Methods:

• Developed ReleAsing Factor Tethered Editing of RNA (RAFTER) by combining an RNA editing enzyme with a translation releasing factor.
• Utilized RAFTER to selectively label RNAs that have undergone successful translation.
• Compared RAFTER results with established methods like Ribo-seq and Polysome-seq.

Main Results:

• RAFTER accurately estimates translation levels, showing strong correlation with Ribo-seq and Polysome-seq.
• The RAFTER workflow is significantly simplified compared to existing methods.
• RAFTER is suitable for single-cell analysis of mRNA translation.

Conclusions:

• RAFTER provides a more accurate and simplified approach to measuring mRNA translation efficiency.
• This technique overcomes limitations of previous methods by focusing on successfully translated RNAs.
• RAFTER opens new possibilities for studying translation dynamics at the single-cell level.