Eukaryotic translation initiation factor 3 (eIF3) is a multi-subunit complex that promotes ribosome recruitment and messenger RNA (mRNA) selection. Here, we show that its eIF3g subunit, along with the binding partner eIF3i, mediates transcript-specific translation under mild heat stress through direct RNA binding. First, SELEX experiments identified a short GUCG-centered motif preferentially recognized by eIF3g, suggesting a sequence-specific binding preference. Next, ribosome profiling of yeast eIF3i mutant revealed that mRNAs containing GUCG motifs in their 5' coding regions exhibit elevated ribosome occupancy in a manner dependent on eIF3g/eIF3i module. A subset of SELEX-identified motifs, collectively termed the GUCG box, was found enriched in the 5'-terminal coding region of the regulated mRNAs. Reporter assays confirmed that these 5'-terminal coding regions are sufficient to drive heat-induced translation. Mutational analyses and biolayer interferometry demonstrated that disruption of the GUCG motif impairs eIF3g binding and diminishes translational induction. Moreover, GUCG motifs are periodically distributed across coding sequences and enriched near start codons, consistent with their role in stabilizing initiating ribosomes. Overall, this study establishes the GUCG box as a bona fide eIF3g-binding motif and validates its functional importance in vivo. These findings provide new insight into how eIF3 mediates stress-adaptive translation through sequence-specific RNA recognition.
© The Author(s) 2026. Published by Oxford University Press.