Abstract
Canonical cap-dependent initiation requires the EIF4F complex, composed of the scaffold EIF4G1, the cap-binding protein EIF4E, and the RNA helicase EIF4A1. Using an inducible degron to acutely deplete EIF4G1, we observed a marked loss of heavy polysomes with a reciprocal increase in monosomes. EIF4G1 depletion also activated initiation-specific ribosome-associated quality control (iRQC), evidenced by RPS3 mono-ubiquitination enriched on 80S monosomes consistent with stalling at start-site commitment. We combined EIF4G1 depletion with polysome RNA-seq to quantify transcript-specific polysome association (PA). This identified mRNAs that were most sensitive or resistant to EIF4G1 loss. Transcript-wide features across the 5′UTR, CDS, and 3′UTR differed between these groups, with transcript length emerging as a unifying theme. We tested whether the CDS GC content was affected by EIF4G1 depletion using a synthetic reporter assay: increasing CDS GC content rendered reporters more resistant to EIF4G1 loss. Together, these results indicate that whole-transcript architecture beyond the 5′UTR shapes apparent EIF4G1 requirement, and that removing EIF4G1 triggers iRQC in a manner consistent with impaired initiation.