Selective Translation Complex Profiling Reveals Staged Initiation and Co-translational Assembly of Initiation Factor...

Area of Science:

• Molecular Biology
• Gene Expression Regulation
• Cellular Biology

Background:

• Translational control, particularly initiation, is crucial for gene expression regulation.
• Existing technologies like ribosome profiling have limitations in capturing specific initiation events.
• Advancements are needed to fully understand the complexities of translational control.

Purpose of the Study:

• To adapt and validate yeast translation complex profile sequencing (TCP-seq) for mammalian cells.
• To investigate the behavior and dynamics of 40S subunits and initiation factors during translation initiation.
• To provide novel insights into mRNA translational control mechanisms.

Main Methods:

• Modification and adaptation of yeast TCP-seq for mammalian cells (human TCP-seq).
• Development of selective TCP-seq (Sel-TCP-seq) for capturing initiation factors with 40S subunits and 80S ribosomes.
• Analysis of 40S and 80S subunit footprints and associated initiation factor complexes.

Main Results:

• Human TCP-seq demonstrated conserved distribution of 40S subunits across 5' untranslated regions (5' UTRs) in mammalian and yeast cells.
• Sel-TCP-seq revealed that eIF2 and eIF3 travel with scanning 40S subunits and dissociate upon AUG recognition, with some eIF3 remaining during early elongation.
• Identification of four initiating 48S conformational intermediates and insights into ATF4 and GCN4 mRNA translational control.

Conclusions:

• TCP-seq is a valuable tool for studying translational control in mammalian systems.
• The dynamics of initiation factors provide a detailed view of the translation initiation process.
• This work enhances our understanding of gene expression regulation at the translational level.