MCTS2 and distinct eIF2D roles in uORF-dependent translation regulation revealed by in vitro re-initiation assays

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• Upstream open reading frames (uORFs) can negatively impact protein biosynthesis by initiating translation.
• Ribosome re-initiation after uORF termination is a regulatory mechanism, but associated factors are not fully understood.
• The MCTS1-DENR complex is known to participate in specific uORF re-initiation events.

Purpose of the Study:

• To investigate novel trans-acting factors and uORF features influencing translation re-initiation.
• To establish and validate a cell-free system for studying re-initiation.
• To differentiate the roles of MCTS1-DENR and its homolog eIF2D in gene regulation.

Main Methods:

• Development of a cell-free re-initiation assay using HeLa cell lysates.
• Comparison of in vivo and in vitro re-initiation using reporter constructs.
• Ribosome profiling in cells to assess gene deregulation upon factor knockdown.
• Identification of protein-protein interactions using the cell-free system.

Main Results:

• Validated MCTS1-DENR-dependent re-initiation in the cell-free system.
• Demonstrated that eIF2D knockdown causes gene deregulation independent of uORF translation.
• Identified MCTS2, a retrogene product, as a DENR partner that promotes re-initiation.
• Linked MCTS2's role to clinical variations observed with DENR and MCTS1 mutations.

Conclusions:

• The study elucidates novel mechanisms and factors governing translation re-initiation at uORFs.
• MCTS2 emerges as a key player in re-initiation, potentially explaining clinical observations.
• Distinguishes the regulatory roles of MCTS1-DENR and eIF2D in gene expression.