MCTS2 and distinct eIF2D roles in uORF-dependent translation regulation revealed by in vitro re-initiation assays

Romane Meurs ¹, Mara De Matos ¹, Adrian Bothe ², Nicolas Guex ³, Tobias Weber ⁴, Aurelio A Teleman ⁴, Nenad Ban ², David Gatfield ⁵

Affiliations Expand

• PMID: 39748120
• DOI: 10.1038/s44318-024-00347-3

Free article

Abstract

Ribosomes scanning from the mRNA 5′ cap to the start codon may initiate at upstream open reading frames (uORFs), decreasing protein biosynthesis. Termination at a uORF can lead to re-initiation, where 40S subunits resume scanning and initiate another translation event downstream. The noncanonical translation factors MCTS1-DENR participate in re-initiation at specific uORFs, but knowledge of other trans-acting factors or uORF features influencing re-initiation is limited. Here, we establish a cell-free re-initiation assay using HeLa lysates to address this question. Comparing in vivo and in vitro re-initiation on uORF-containing reporters, we validate MCTS1-DENR-dependent re-initiation in vitro. Using this system and ribosome profiling in cells, we found that knockdown of the MCTS1-DENR homolog eIF2D causes widespread gene deregulation unrelated to uORF translation, and thus distinct to MCTS1-DENR-dependent re-initiation regulation. Additionally, we identified MCTS2, encoded by an Mcts1 retrogene, as a DENR partner promoting re-initiation in vitro, providing a plausible explanation for clinical differences associated with DENR vs. MCTS1 mutations in humans.

PPARβ/δ upregulates the insulin receptor β subunit in skeletal muscle by reducing lysosomal activity and EphB4 levels

Jue-Rui Wang ^# 1 2 3, Javier Jurado-Aguilar ^# 1 2 3, Emma Barroso ^1 2 3, Ricardo Rodríguez-Calvo ^2 4, Antoni Camins ^1 5 6, Walter Wahli ^7 8, Xavier Palomer ^1 2 3, Manuel Vázquez-Carrera ^{9 10 11 12}

• PMID: 39696437
• PMCID: PMC11656877
• DOI: 10.1186/s12964-024-01972-5

Abstract

Background: The increased degradation of the insulin receptor β subunit (InsRβ) in lysosomes contributes to the development of insulin resistance and type 2 diabetes mellitus. Endoplasmic reticulum (ER) stress contributes to insulin resistance through several mechanisms, including the reduction of InsRβ levels. Here, we examined how peroxisome proliferator-activated receptor (PPAR)β/δ regulates InsRβ levels in mouse skeletal muscle and C2C12 myotubes exposed to the ER stressor tunicamycin.

Methods: Wild-type (WT) and Ppard^-/- mice, WT mice treated with vehicle or the PPARβ/δ agonist GW501516, and C2C12 myotubes treated with the ER stressor tunicamycin or different activators or inhibitors were used.

Results: Ppard^-/- mice displayed reduced InsRβ protein levels in their skeletal muscle compared to wild-type (WT) mice, while the PPARβ/δ agonist GW501516 increased its levels in WT mice. Co-incubation of tunicamycin-exposed C2C12 myotubes with GW501516 partially reversed the decrease in InsRβ protein levels, attenuating both ER stress and the increase in lysosomal activity. In addition, the protein levels of the tyrosine kinase ephrin receptor B4 (EphB4), which binds to the InsRβ and facilitates its endocytosis and degradation in lysosomes, were increased in the skeletal muscle of Ppard^-/- mice, with GW501516 reducing its levels in the skeletal muscle of WT mice.

Conclusions: Overall, these findings reveal that PPARβ/δ activation increases InsRβ levels by alleviating ER stress and lysosomal degradation.

Lipid sensing by PPARα: Role in controlling hepatocyte gene regulatory networks and the metabolic response to fasting

Anne Fougerat ¹, Justine Bruse ², Arnaud Polizzi ², Alexandra Montagner ³, Hervé Guillou ², Walter Wahli ⁴

 Epub 2024 Nov 7.

• PMID: 39521352
• DOI: 10.1016/j.plipres.2024.101303

Free article

Abstract

Peroxisome proliferator-activated receptors (PPARs) constitute a small family of three nuclear receptors that act as lipid sensors, and thereby regulate the transcription of genes having key roles in hepatic and whole-body energy homeostasis, and in other processes (e.g., inflammation), which have far-reaching health consequences. Peroxisome proliferator-activated receptor isotype α (PPARα) is expressed in oxidative tissues, particularly in the liver, carrying out critical functions during the adaptive fasting response. Advanced omics technologies have provided insight into the vast complexity of the regulation of PPAR expression and activity, as well as their downstream effects on the physiology of the liver and its associated metabolic organs. Here, we provide an overview of the gene regulatory networks controlled by PPARα in the liver in response to fasting. We discuss impacts on liver metabolism, the systemic repercussions and benefits of PPARα-regulated ketogenesis and production of fibroblast growth factor 21 (FGF21), a fasting- and stress-inducible metabolic hormone. We also highlight current challenges in using novel methods to further improve our knowledge of PPARα in health and disease.