Poly(U)‐specific endoribonuclease ENDOU promotes translation of human CHOP mRNA by releasing uORF element‐mediated inhibition

Upstream open reading frames (uORFs) are known to negatively affect translation of the downstream ORF. The regulatory proteins involved in relieving this inhibition are however poorly characterized. In response to cellular stress, eIF2α phosphorylation leads to an inhibition of global protein synthesis, while translation of specific factors such as CHOP is induced. We analyzed a 105‐nt inhibitory uORF in the transcript of human CHOP ( huORF chop ) and found that overexpression of the zebrafish or human ENDOU poly(U)‐endoribonuclease (Endouc or ENDOU‐1, respectively) increases CHOP mRNA translation also in the absence of stress. We also found that Endouc/ENDOU‐1 binds and cleaves the huORF chop transcript at position 80G‐81U, which induces CHOP translation independently of phosphorylated eIF2α. However, both ENDOU and phospho‐eIF2α are nonetheless required for maximal translation of CHOP mRNA. Increased levels of ENDOU shift a huORF chop reporter as well as endogenous CHOP transcripts from the monosome to polysome fraction, indicating an increase in translation. Furthermore, we found that the uncapped truncated huORF chop ‐69‐105‐nt transcript contains an internal ribosome entry site (IRES), facilitating translation of the cleaved transcript. Therefore, we propose a model where ENDOU‐mediated transcript cleavage positively regulates CHOP translation resulting in increased CHOP protein levels upon stress. Specifically, CHOP transcript cleavage changes the configuration of huORF chop thereby releasing its inhibition and allowing the stalled ribosomes to resume translation of the downstream ORF. SYNOPSIS Heat or ER stress trigger a stress response involving eIF2α phosphorylation and induction of the transcription factor CHOP. Here, stress‐induced poly(U)‐specific endoribonuclease is found to specifically cleave CHOP mRNA, thereby releasing a uORF‐associated inhibitory structure and allowing its selective translation. Zebrafish Endouc and its human orthologue ENDOU positively regulate CHOP translation independently of phosphorylated eIF2α. Both ENDOU and phospho‐eIF2α are required for maximal translation of CHOP upon heat shock or ER stress. ENDOU binds the 105‐nt upstream uORF of the human CHOP transcript and cleaves it at position 80G‐81U. The truncated huORFchop‐81‐105 transcript does not contain IRES activity, while uncapped truncated huORFchop‐69‐105 transcript does. Graphical Abstract Cleavage of CHOP transcripts by zebrafish Endouc and its human ortholog