eIF3d controls the persistent integrated stress response.

Cells respond to intrinsic and extrinsic stresses by reducing global protein synthesis and activating gene programs necessary for survival. Here, we show that the integrated stress response (ISR) is driven by the non-canonical cap-binding protein eIF3d that acts as a critical effector to control core stress response orchestrators, the translation factor eIF2α and the transcription factor ATF4. We find that during persistent stress, eIF3d activates the translation of the kinase GCN2, inducing eIF2α phosphorylation and inhibiting general protein synthesis. In parallel, eIF3d upregulates the m6A demethylase ALKBH5 to drive 5' UTR-specific demethylation of stress response genes, including ATF4. Ultimately, this cascade converges on ATF4 expression by increasing mRNA engagement of translation machinery and enhancing ribosome bypass of upstream open reading frames (uORFs). Our results reveal that eIF3d acts in a life-or-death decision point during chronic stress and uncover a synergistic signaling mechanism in which translational cascades complement transcriptional amplification to control essential cellular processes.

RNA polymerase II transcription attenuation at the yeast DNA repair gene DEF1 is biologically significant and dependent on the Hrp1 RNA-recognition motif.

Premature transcription termination (i.e. attenuation) is a potent gene regulatory mechanism that represses mRNA synthesis. Attenuation of RNA polymerase II is more prevalent than once appreciated, targeting 10-15% of mRNA genes in yeast through higher eukaryotes, but its significance and mechanism remain obscure. In the yeast Saccharomyces cerevisiae, polymerase II attenuation was initially shown to rely on Nrd1-Nab3-Sen1 termination, but more recently our laboratory characterized a hybrid termination pathway involving Hrp1, an RNA-binding protein in the 3'-end cleavage factor. One of the hybrid attenuation gene targets is DEF1, which encodes a repair protein that promotes degradation of polymerase II stalled at DNA lesions. In this study, we characterized the chromosomal DEF1 attenuator and the functional role of Hrp1. DEF1 attenuator mutants overexpressed Def1 mRNA and protein, exacerbated polymerase II degradation, and hindered cell growth, supporting a biologically significant DEF1 attenuator function. Using an auxin-induced Hrp1 depletion system, we identified new Hrp1-dependent attenuators in MNR2, SNG1, and RAD3 genes. An hrp1-5 mutant (L205S) known to impair binding to cleavage factor protein Rna14 also disrupted attenuation, but surprisingly no widespread defect was observed for an hrp1-1 mutant (K160E) located in the RNA-recognition motif. We designed a new RNA recognition motif mutant (hrp1-F162W) that altered a highly conserved residue and was lethal in single copy. In a heterozygous strain, hrp1-F162W exhibited dominant-negative readthrough defects at several gene attenuators. Overall, our results expand the hybrid RNA polymerase II termination pathway, confirming that Hrp1-dependent attenuation controls multiple yeast genes and may function through binding cleavage factor proteins and/or RNA.