eIF2D promotes 40S ribosomal subunit recycling during intrinsic ribosome destabilization

Although eukaryotic initiation factor 2D (eIF2D) is implicated in translation initiation, re-initiation, and ribosome recycling, its precise role remains unclear. Here, we show that eIF2D promotes 40S ribosome recycling during intrinsic ribosome destabilization (IRD), a process in which ribosomes stochastically destabilize while translating proteins with consecutive acidic amino acids at their NH2-terminus. Unrecycled 40S ribosomes accumulate in eIF2D-deficient cells, leading to 80S ribosome stalling. Selective translation complex profiling (TCP-seq) reveals that eIF2D preferentially associates with IRD-prone regions. The winged-helix (WH) domain, unique to eIF2D but absent in MCTS1–DENR, enhances its binding to 40S subunits, but likely clashes with ABCE1 during stop codon-associated recycling. Loss of eIF2D reduces the expression of IRD-inducing proteins, including splicing factors. Together, these findings define a previously unappreciated role for eIF2D in 40S recycling and clarify its mechanistic divergence from the MCTS1–DENR complex. Overall design: Translatome and transcriptome analysis of eIF2D-deficient HEK293T cells