Capturing Translation in Action with Protein Synthesis Profiling.

Area of Science:

• Molecular Biology
• Gene Expression Regulation
• Biochemistry

Background:

• Gene expression is controlled at the translation level, linking genetic sequences to functional proteins.
• Disruptions in translation are implicated in numerous diseases, necessitating methods to identify actively translated transcripts.
• Current methods like ribosome profiling have limitations, including laborious procedures and loss of information on mRNA isoforms and modifications.

Purpose of the Study:

• To introduce Protein Synthesis Profiling (PSP), a novel proximity-labeling strategy for transcriptome-wide identification of actively translated mRNAs.
• To overcome the limitations of existing methods by enabling translation analysis without ribosome isolation and preserving full-length transcript information.

Main Methods:

• PSP utilizes a fusion of the APEX2 enzyme with elongation factor eEF2 to selectively tag mRNAs engaged in translation.
• The method employs proximity labeling, catalyzed by the APEX2-eEF2 fusion, to label mRNAs associated with translating ribosomes.
• The approach was applied in *Saccharomyces cerevisiae* to profile actively translated transcripts.

Main Results:

• PSP successfully captured condition-specific translational programs and recapitulated known stress responses in yeast.
• The method identified a broader repertoire of regulated genes compared to existing techniques.
• PSP preserves full-length transcript features, enabling isoform-aware analysis and revealing comprehensive translational regulation.

Conclusions:

• Protein Synthesis Profiling (PSP) provides a powerful, scalable, and adaptable platform for studying translational regulation.
• PSP overcomes key limitations of ribosome profiling, offering a versatile tool for dissecting mRNA translation in health and disease.
• The method enhances the understanding of gene expression control at the translational level.