Streamlined and sensitive mono- and di-ribosome profiling in yeast and human cells

Area of Science:

• Molecular Biology
• Genomics
• Biochemistry

Background:

• Ribosome profiling is a powerful technique for surveying translation and ribosome occupancy across the transcriptome.
• Current methods for generating ribosome footprints are technically demanding and prone to biases.
• These biases can distort the accurate representation of physiological ribosome occupancy.

Purpose of the Study:

• To develop an optimized ribosome profiling method that overcomes technical challenges and reduces bias.
• To improve the accuracy and richness of translatome profiling.
• To provide a more reliable tool for studying translation regulation and dynamics.

Main Methods:

• Utilized P1 nuclease instead of RNase I for generating ribosome footprints.
• Replaced traditional RNA ligation with an ordered two-template relay protocol for library preparation.
• Incorporated adaptors via reverse transcription in a single-tube protocol.

Main Results:

• The new method significantly reduced sequence bias compared to existing protocols.
• Enhanced enrichment of ribosome footprints relative to ribosomal RNA was observed.
• P1 nuclease preserved distinct ribosome complexes, offering insights into translation initiation, stalling, and termination in yeast and human systems.

Conclusions:

• The optimized ribosome footprint generation and capture methods offer a richer translatome profile.
• This streamlined approach requires lower input and presents fewer technical challenges.
• The improved method facilitates a more comprehensive understanding of translational regulation.