Protein synthesis plays a major role in homeostasis and when dysregulated leads to various pathologies including cancer. To this end, imbalanced expression of eukaryotic translation initiation factors (eIFs) is not only a consequence but also a driver of neoplastic growth. eIF3 is the largest, multi-subunit translation initiation complex with a modular assembly, where aberrant expression of one subunit generates only partially functional subcomplexes. To comprehensively study the effects of eIF3 remodeling, we contrasted the impact of eIF3d, eIF3e or eIF3h depletion on the translatome of HeLa cells using Ribo-seq. Depletion of eIF3d or eIF3e, but not eIF3h reduced the levels of multiple components of the MAPK signaling pathways. Surprisingly, however, depletion of all three eIF3 subunits increased MAPK/ERK pathway activity. Depletion of eIF3e and partially eIF3d also increased translation of TOP mRNAs that encode mainly ribosomal proteins and other components of the translational machinery. Moreover, alterations in eIF3 subunit stoichiometry were often associated with changes in translation of mRNAs containing short uORFs, as in the case of the proto-oncogene MDM2 and the transcription factor ATF4. Collectively, perturbations in eIF3 subunit stoichiometry exert specific effect on the translatome comprising signaling and stress-related transcripts with complex 5' UTRs that are implicated in homeostatic adaptation to stress and cancer.

• Keywords
• MAPK pathway, eIF3, genetics, genomics, human, ribosomal proteins, ribosome, translation, translational control,
• MeSH
• Eukaryotic Initiation Factor-3 * metabolism   genetics   MeSH
• HeLa Cells MeSH
• Humans MeSH
• MAP Kinase Signaling System * MeSH
• Protein Biosynthesis MeSH
• Proto-Oncogene Mas * MeSH
• Ribosomal Proteins * metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Eukaryotic Initiation Factor-3 * MeSH
• MAS1 protein, human MeSH Browser
• Proto-Oncogene Mas * MeSH
• Ribosomal Proteins * MeSH

Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation (REI) at short upstream open reading frames (uORFs) harboring penultimate codons that confer heightened dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited REI at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on REI at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) REI. We found that the Tma proteins generally impede REI at native uORF4 and its variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on REI at native uORF1 and equipping it with Tma-hyperdependent penultimate codons generally did not confer Tma-dependent REI; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the REI potential of the uORF and penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.

• MeSH
• Peptide Chain Initiation, Translational MeSH
• Ribosome Subunits, Small, Eukaryotic * metabolism   genetics   MeSH
• RNA, Messenger * metabolism   genetics   MeSH
• Open Reading Frames * MeSH
• Protein Biosynthesis MeSH
• Saccharomyces cerevisiae Proteins * metabolism   genetics   MeSH
• Saccharomyces cerevisiae * genetics   metabolism   MeSH
• Basic-Leucine Zipper Transcription Factors * metabolism   genetics   MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• GCN4 protein, S cerevisiae MeSH Browser
• RNA, Messenger * MeSH
• Saccharomyces cerevisiae Proteins * MeSH
• Basic-Leucine Zipper Transcription Factors * MeSH

Activating transcription factor 4 (ATF4) is a master transcriptional regulator of the integrated stress response, leading cells toward adaptation or death. ATF4's induction under stress was thought to be due to delayed translation reinitiation, where the reinitiation-permissive upstream open reading frame 1 (uORF1) plays a key role. Accumulating evidence challenging this mechanism as the sole source of ATF4 translation control prompted us to investigate additional regulatory routes. We identified a highly conserved stem-loop in the uORF2/ATF4 overlap, immediately preceded by a near-cognate CUG, which introduces another layer of regulation in the form of ribosome queuing. These elements explain how the inhibitory uORF2 can be translated under stress, confirming prior observations but contradicting the original regulatory model. We also identified two highly conserved, potentially modified adenines performing antagonistic roles. Finally, we demonstrated that the canonical ATF4 translation start site is substantially leaky scanned. Thus, ATF4's translational control is more complex than originally described, underpinning its key role in diverse biological processes.

• Keywords
• ATF4, CP: Molecular biology, integrated stress response, ribosome, ribosome queuing, translation reinitiation, translational control, unfolded protein response,
• MeSH
• Stress, Physiological MeSH
• HEK293 Cells MeSH
• Humans MeSH
• Open Reading Frames * genetics   MeSH
• Protein Biosynthesis * MeSH
• Ribosomes * metabolism   MeSH
• Base Sequence MeSH
• Activating Transcription Factor 4 * metabolism   genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, N.I.H., Extramural MeSH
• Names of Substances
• ATF4 protein, human MeSH Browser
• Activating Transcription Factor 4 * MeSH

Recycling of 40S ribosomal subunits following translation termination, entailing release of deacylated tRNA and dissociation of the empty 40S subunit from mRNA, involves yeast Tma20/Tma22 heterodimer and Tma64, counterparts of mammalian MCTS1/DENR and eIF2D. MCTS1/DENR enhance reinitiation at short upstream open reading frames (uORFs) harboring penultimate codons that confer dependence on these factors in bulk 40S recycling. Tma factors, by contrast, inhibited reinitiation at particular uORFs in extracts; however, their roles at regulatory uORFs in vivo were unknown. We examined effects of eliminating Tma proteins on reinitiation at regulatory uORFs mediating translational control of GCN4 optimized for either promoting (uORF1) or preventing (uORF4) reinitiation. We found that the Tma proteins generally impede reinitiation at native uORF4 and uORF4 variants equipped with various penultimate codons regardless of their Tma-dependence in bulk recycling. The Tma factors have no effect on reinitiation at native uORF1, and equipping uORF1 with Tma-dependent penultimate codons generally did not confer Tma-dependent reinitiation; nor did converting the uORFs to AUG-stop elements. Thus, effects of the Tma proteins vary depending on the reinitiation potential of the uORF and the penultimate codon, but unlike in mammals, are not principally dictated by the Tma-dependence of the codon in bulk 40S recycling.

• Keywords
• DENR, MCTS1, Tma, eIF2D, reinitiation, ribosome recycling,
• Publication type
• Journal Article MeSH
• Preprint MeSH

Multiple aspects of mRNA translation are subject to regulation. Here we present a ribosome footprinting protocol to determine the location and composition of 40S and 80S ribosome complexes on endogenous mRNAs transcriptome-wide in vivo in yeast and mammalian cells. We present an extension of the translation complex profiling (TCP-seq) protocol, originally developed in yeast, by including an immunoprecipitation step to assay the location of both 40S and 80S ribosome complexes containing proteins of interest. This yields information on where along mRNAs the ribosome-bound protein of interest joins the ribosome to act, and where it leaves again, thereby monitoring the sequential steps of translation and the roles of various translation factors therein. Rapid fixation of live cells ensures the integrity of all translation complexes bound to mRNA at native positions. Two procedures are described, differing mainly in the fixation conditions and the library preparation. Depending on the research question, either procedure offers advantages. Execution of a Sel-TCP-seq experiment takes 5-10 working days, and initial data analysis can be completed within 2 days.

• MeSH
• RNA, Messenger genetics   MeSH
• Protein Biosynthesis * MeSH
• Ribosomes genetics   MeSH
• Saccharomyces cerevisiae * genetics   MeSH
• Mammals genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Review MeSH
• Names of Substances
• RNA, Messenger MeSH

Translation reinitiation is a gene-specific translational control mechanism. It is characterized by the ability of short upstream ORFs to prevent full ribosomal recycling and allow the post-termination 40S subunit to resume traversing downstream for the next initiation event. It is well known that variable transcript-specific features of various uORFs and their prospective interactions with initiation factors lend them an unequivocal regulatory potential. Here, we investigated the proposed role of the major initiation scaffold protein eIF4G in reinitiation and its prospective interactions with uORF's cis-acting features in yeast. In analogy to the eIF3 complex, we found that eIF4G and eIF4A but not eIF4E (all constituting the eIF4F complex) are preferentially retained on ribosomes elongating and terminating on reinitiation-permissive uORFs. The loss of the eIF4G contact with eIF4A specifically increased this retention and, as a result, increased the efficiency of reinitiation on downstream initiation codons. Combining the eIF4A-binding mutation with that affecting the integrity of the eIF4G1-RNA2-binding domain eliminated this specificity and produced epistatic interaction with a mutation in one specific cis-acting feature. We conclude that similar to humans, eIF4G is retained on ribosomes elongating uORFs to control reinitiation also in yeast.

• MeSH
• DEAD-box RNA Helicases genetics   MeSH
• Eukaryotic Initiation Factor-3 genetics   MeSH
• Eukaryotic Initiation Factor-4E genetics   MeSH
• Eukaryotic Initiation Factor-4G genetics   MeSH
• Peptide Chain Initiation, Translational genetics   MeSH
• Codon, Initiator genetics   MeSH
• Humans MeSH
• Open Reading Frames genetics   MeSH
• Protein Biosynthesis genetics   MeSH
• Ribosomes genetics   MeSH
• Saccharomyces cerevisiae Proteins genetics   MeSH
• Saccharomyces cerevisiae genetics   MeSH
• Basic-Leucine Zipper Transcription Factors genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DEAD-box RNA Helicases MeSH
• Eukaryotic Initiation Factor-3 MeSH
• Eukaryotic Initiation Factor-4E MeSH
• Eukaryotic Initiation Factor-4G MeSH
• Codon, Initiator MeSH
• PRP2 protein, S cerevisiae MeSH Browser
• Saccharomyces cerevisiae Proteins MeSH
• TIF4631 protein, S cerevisiae MeSH Browser
• Basic-Leucine Zipper Transcription Factors MeSH