DAP5 enables translation re-initiation on structured messenger RNAs

Abstract

SummaryHalf 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 selectively required for re-initiation at the main CDS following uORF translation. Using ribosome profiling and luciferase-based reporters coupled with mutational analysis we show that DAP5-mediated re-initiation 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 re-initiation by unrecycled post-termination 40S subunits. 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.

Loss-of-function cancer-associated mutations in the EIF4G2 non-canonical translation initiation factor

“…Various established EIF4G2 targets that represent different translation initiation mechanisms were assessed. These included BCL2, representing IRES-directed targets (Marash et al, 2008;Yoffe et al, 2016), and ROCK1 and WNK1, which contain cap-dependent uORFs that drive re-initiation of the downstream CDS in an EIF4G2-dependent mechanism (David et al, 2022;Weber et al, 2022). We first confirmed the EIF4G2-dependency of all three proteins in EIF4G2 knock-out (KO) 293T cells, generated by the CRISPR-Cas9 system (Fig EV3A).…”

“…2x10 5 293T-EIF4G2KO cells were seeded in 6 well plates and transfected with 5 μg empty pcDNA3, pcDNA3-FLAG-EIF4G2_WT, pcDNA3-FLAG-EIF4G2_R165C, pcDNA3-FLAG-EIF4G2_R178Q, pcDNA3-FLAG-EIF4G2_R295C, pcDNA3-FLAG-EIF4G2_R714H or pcDNA3-FLAG-EIF4G2_N785K together with 1 μg pHP-IRES-BCL2-F-LUC plasmid (Liberman et al, 2015;Yoffe et al, 2016) with 1 μg pCIneo-R-LUC plasmid as control, or alternatively, 1 μg pCIneo-WNK1-R-LUC or pCIneo-ROCK1-R-LUC with 1μg pEGFP-N3-F-LUC plasmids (kindly gifted from the Igreja lab (Weber et al, 2022), Max Planck Institute, Germany), using Lipofectamine 2000 (Invitrogen) according to the manufacturer's recommendations.…”

“…Unlike EIF4G1, EIF4G2 lacks the PABP and EIF4E binding sites, and therefore instead participates in several cap-independent modes of translation initiation, such as those utilizing internal ribosome entry sites [IRES] (Henis-Korenblit et al, 2002;Hundsdoerfer et al, 2005;Lewis et al, 2008;Liberman et al, 2015;Marash & Kimchi, 2005;Warnakulasuriyarachchi et al, 2004) or capindependent translation enhancers [CITE] (Haizel et al, 2020), and N6methyladenosine [m6A] driven translation (Yang et al, 2017). Additionally, EIF4G2 has been shown to promote read-through of 5' upstream open reading frames [uORFs] (Smirnova et al, 2022) and/or re-initiation of the main coding sequence [CDS] following cap-dependent translation of these uORFs (Weber et al, 2022). It can also mediate a non-canonical cap-dependent method of initiation by binding EIF3D (de la Parra et al, 2018a;Volta et al, 2021), which replaces EIF4E as the mRNA 5' cap interactor (Lee et al, 2016).…”

RNA binding protein PRRC2B mediates translation of specific mRNAs and regulates cell cycle progression

“…Although CU- and GA-rich motifs are found throughout mature mRNAs (in 5’ UTR, CDS, and 3’ UTR), PRRC2B predominately binds the motif sequence near the translation initiation sites where it interacts with translation initiation factors eIF4G2 and eIF3. Despite the fact that eIF4G2 has been accounted for transcript selectivity (23,39), interacting with it hardly affects PRRC2B binding since the PAR-CLIP for P2, the 751-1500 amino acid-containing fragment of PRRC2B not interacting with eIF4G2 or eIF3, exhibited similar binding profile and target sequences as the full-length PRRC2B. Based on this, we conclude that P2 region of PRRC2B dictates at least some degree of transcript selectivity in an eIF4G2- and eIF3-independent manner.…”

Multifunctional RNA-binding proteins influence mRNA abundance and translational efficiency of distinct sets of target genes

“…These significant targets, for instance, included the candidate tumor suppressor and ubiquitous splicing regulator RBM5 [ 37 ], which we identified as a cardiac mRNA-RBP influencing mRNA abundance of at least 138 correlating targets (p adj = 2.83 × 10 −5 ; Glass’ Δ = 27.2). Reassuringly, our strategy validated known TE-RBPs such as the eukaryotic translation initiation factor EIF4G2, whose expression dynamics could be associated with target gene translational efficiencies of at least 235 correlating targets expressed in the human heart (p adj = 5.26 × 10 −5 ; Glass’ Δ = 6.3) [ 38 , 39 ] ( Fig 1B ). Importantly, we could replicate our calculations for 25 out of 37 depicted TE-RBPs in an independent, though smaller cohort of primary cardiac fibroblast translatomes (n = 20; [ 30 ])—a system previously explored to identify RBPs with key roles in cardiac fibrosis ( Fig 1C ).…”