DAP5 enables main ORF translation on mRNAs with structured and uORF-containing 5′ leaders

Abstract

Half of mammalian transcripts contain short upstream open reading frames (uORFs) that potentially regulate translation of the downstream coding sequence (CDS). The molecular mechanisms governing these events remain poorly understood. Here, we find that the non-canonical initiation factor Death-associated protein 5 (DAP5 or eIF4G2) is required for translation initiation on select transcripts. Using ribosome profiling and luciferase-based reporters coupled with mutational analysis we show that DAP5-mediated translation occurs on messenger RNAs (mRNAs) with long, structure-prone 5′ leader sequences and persistent uORF translation. These mRNAs preferentially code for signalling factors such as kinases and phosphatases. We also report that cap/eIF4F- and eIF4A-dependent recruitment of DAP5 to the mRNA facilitates main CDS, but not uORF, translation suggesting a role for DAP5 in translation re-initiation. Our study reveals important mechanistic insights into how a non-canonical translation initiation factor involved in stem cell fate shapes the synthesis of specific signalling factors.

Ribosome rescue factor PELOTA modulates translation start site choice and protein isoform levels of transcription factor C/EBPα

“…The CEBPA 5’ UTR is highly structured in addition to containing a uORF, making it a prime target for DAP5 dependence as well 59 . While the molecular details of DAP5 function are not clear, its general role in downstream CDS translation in the context of structured 5’ UTRs is consistent with its importance for short isoform translation on CEBPA .…”

“…The shift towards long isoform production in these knockdowns is consistent with a defect in translation reinitiation at the short isoform start codon. Depletion of the eIF4G paralog eIF4G2/DAP5, also implicated in reinitiation 59,60 , caused a strong increase in green fluorescence as well (Figure S3A). Interestingly, depletion of the initiation factor eIF2α, encoded by EIF2S1, actually decreased the green/red ratio, in contrast with the observation that increasing eIF2α availability causes a shift towards short isoform production 14 .…”

Specific mechanisms of translation initiation in higher eukaryotes: the eIF4G2 story

“…Binding to eIF2 is important for the mechanics of eIF4G2. Swapping the W2 domains with eIF4G1 results in eIF4G2 malfunction ( Weber et al 2022 ) and a point mutation (E862K) that prevents eIF2 binding inactivates eIF4G2 ( Bryant et al 2018 ). The eIF4G2–eIF2β interaction is correlated with MEK1/2 or CDK1 activation and eIF4G2 phosphorylation (T508), and treatment with MEK1/2 inhibitors completely abolishes both binding and phosphorylation.…”

“…This may be the case for DSCR1.4 mRNA, which only marginally depends on m 7 G-cap and requires eIF4G2 for its expression ( Seo et al 2019 ). In fact, many mRNAs have been shown to interact with eIF4G2 ( Marash et al 2008 ; Weingarten-Gabbay et al 2014 ; Yoffe et al 2016 ; Seo et al 2019 ; Haizel et al 2020 ; Weber et al 2022 ; David et al 2022 ). In most cases, the binding was linked to eIF4G2-driven internal initiation, but it is not possible to unambiguously interpret the data as a result of the use of error-prone methodology.…”

“…The majority of validated eIF4G2-dependent reporter mRNAs show rather high cap-dependencies ( Smirnova et al 2019 , 2022 ) and are sensitive to eIF4E inactivation caused by PP242-mediated inhibition of mTOR kinase or overexpression of 4E-BP1 ( Weber et al 2022 ). Similarly, eIF4G2 knockdown does not affect the response of eIF4G2 targets to mTOR inactivation.…”