Poster abstracts
Poster number 4 submitted by Tetyana Lukash
A novel micropeptide suppresses translation of the stress response factor ATF4
Tetyana Lukash (Cell Biology, University of Pittsburgh, School of Medicine), Shyama Nandakumar (Cell Biology, University of Pittsburgh, School of Medicine), Deepika Vasudevan (Cell Biology, University of Pittsburgh, School of Medicine)
Abstract:
The transcription factor ATF4 is a key effector of the integrated stress response (ISR), an evolutionarily conserved pathway that enables eukaryotic cells to adapt to diverse stressors, including ER stress. Upon ISR activation, stress-activated protein kinases phosphorylate the initiator methionine carrier eIF2, broadly suppressing global protein synthesis. This reduces accumulation of misfolded proteins, but, paradoxically, enhances translation of ATF4 due to the specialized architecture of its 5′ leader. The ATF4 5’ leader contains multiple short upstream ORFs (uORFs), with the ultimate uORF sequence overlapping the ATF4-encoding main ORF. Although the number of uORFs varies from species to species, this architecture of the ATF4 mRNA is conserved across metazoa. In humans, uORF1 encodes a tripeptide, while the overlapping uORF2 encodes a 60-amino-acid micropeptide (µPATF4). Under homeostasis, occasional ribosomal reinitiation at uORF2 favors µPATF4 synthesis. Under stress, reduced initiator methionine availability lowers reinitiation at uORF2, allowing ribosomes to initiate instead at the ATF4 start codon. This creates a dynamic trade-off between µPATF4 and ATF4 synthesis: µPATF4 is favored in homeostasis, while ATF4 translation predominates under stress. A role for µPATF4 has never been examined.
Our data show that overexpression of µPATF4 in HeLa cells reduces ATF4 induction in response to ER stress without significantly affecting ATF4 mRNA levels and global translation. To test whether µPATF4 directly regulates ATF4 translation, we employed an ATF4 5’-leader dual-luciferase reporter assay in which the coding sequence of human ATF4 was replaced with Renilla luciferase. Co-transfection of this reporter with µPATF4 showed no induction of luciferase activity in response to ER stress, indicating that µPATF4 acts through the ATF4 5′ leader to specifically regulate its translation. Notably, µPATF4 activity was independent of known regulators of translation reinitiation such as DENR. Polyribosome gradient analysis revealed that µPATF4 is enriched in 40S ribosomal fractions, suggesting a possible role of µPATF4 in regulation of ATF4 translation initiation. Together, these findings uncover a novel modality of translation regulation by a micropeptide.
Keywords: micropeptide, ATF4, translation