Origin of translational control by eIF2α phosphorylation: insights from genome-wide translational profiling studies in fission yeast

Abstract

During amino acid limitation, the protein kinase Gcn2 phosphorylates the α subunit of eIF2, thereby regulating mRNA translation. In yeast Saccharomyces cerevisiae and mammals, eIF2α phosphorylation regulates translation of related transcription factors Gcn4 and Atf4 through upstream open reading frames (uORFs) to activate transcription genome wide. However, mammals encode three more eIF2α kinases activated by distinct stimuli. Did the translational control system involving eIF2α phosphorylation evolve from so simple (as found in yeast S. cerevisiae) to complex (as found in humans)? Recent genome-wide translational profiling studies of amino acid starvation response in the fission yeast Schizosaccharomyces pombe provide an unexpected answer to this question.

Keywords: Evolution; Schizosaccharomyces pombe; Translational control; eIF2α kinase; uORF.

Fig. 1.. Translational control by paired uORFs.

(A) Model of GCN4 translational control. The schematics to the top describe the structure of GCN4 mRNA with boxes indicating uORFs. Table below describes the uORF-dependent delayed re-initiation model for GCN4 translation in unstressed cells (panel 1) and in stressed cells (panel 2). Gray ovals in the schematics represent ribosomes with 40S (smaller oval) and 60S (larger oval) subunits or the subunit alone. Black straight arrows indicate 40S ribosome scanning. Brown rounded arrows indicate ribosome dissociation. See text for details. (B) to (D) uORFs found in the leader regions of GCN4/atf4/cpc1 homologs (B), S. pombe fil1 and gcn5 (C) and fungal hri (D). See Fig. 2 for the classification of species shown. Boxes indicate uORFs (yellow, positive or blue, negative element, respectively) or the main CDS (gray). For dipeptide-coding uORFs, the amino acid sequences (MC, MM and MI) of dipeptides originating from the uORFs are shown below. Overlapping uORF has been considered characteristic of the negative element, as found with Homo sapiens atf1 uORF2. However, this does not appear to be the case in diverse fungi including S. cerevisiae bearing GCN4 uORF4.

Fig. 2.. Conservation and diversity of translational control in fungi.

Left diagram depicts a simplified fungal tree of life with a branch on top representing Metazoa. Divergence time is indicated at branch points (Hoffman et al., 2015; Taylor and Berbee, 2006; Wang et al., 1999): My, million years ago; By, billion years ago. Columns 1–4 indicate the presence (+) or absence (−) of eIF2α My, million yearsHri, 5MP, or Gcn4/Atf4/Cpc1 homolog. ±, Gcn4 homolog is found in L. transversale, but only a handful of members belonging to the phylum Mucoromycota have this homolog. Partially adapted from Fig. 7A of (Chikashige et al., 2020). Identity of the proteins present in the indicated species is described in the legends to this figure, except for L. transversale proteins (Gcn2, XP_021880655; Hri, XP_021881008; 5MP, XP_021884142; and Gcn4, XP_021881329).

Fig. 3.. 5MP phylogenetic tree from diverse eukaryotes.

The tree was generated by MAFFT version 7, with 100 bootstrap replicates on (https://mafft.cbrc.jp/) using sequences obtained from Genbank. Boxes to the right indicate the kingdoms to which the organisms of interest belong to. Bars indicate their subphyla and, for fungi, their classes. Red box, the sole Ascomycota homolog. Bootstrap values are indicated at the nodes. Adapted from Fig. S10B of (Chikashige et al., 2020).

Fig. 4.. Translational profiling methods.

(A) Polysome profiling. Top indicates a typical A₂₅₄ profile of polysomes. Bars indicate boundaries of fractions taken with their numbers indicated in-between. Numbers below the graph indicates the number of translating ribosomes bound to mRNAs in each fraction. Schematics below depicts mRNA (brown line) loaded with different numbers of ribosomes (depicted as ovals as in Fig. 1A) found in each fraction. RNA from each fraction is quantified with DNA microarray (Microarray) or sequenced (RNAseq). The graph below shows the simulated ribosome mass in each fraction using the ribosome numbers as assigned above and mRNA abundance values obtained by microarray hybridization. (B) Ribosome profiling. The flow chart depicts the method of generation and sequencing of ribosome-protected mRNA fragments (RPF, short brown lines) from polysomes (the schematics on top, depicted as in panel A). Bottom graph presents an example of RPF read mapping and shows the protection patters in the 5’ half of fil1 mRNA. Plots are color-coded by three reading frames presented on bottom. Schematics in the middle represent uORF structures and the main CDS also color-coded similarly. Asterisk, the green peak located before uORF1 represents a possible additional uORF initiated by a CUG codon (Duncan et al., 2018). Adapted from Fig. S6A of (Chikashige et al., 2020).

Fig. 5.. Translational control by eIF2α phosphorylation.

(A) Amino acid starvation pathway in S. pombe. Oxi, oxidative stress. Genes on the bottom are the targets of eIF2α-P. Toolkit (cis), the regulatory motifs used for translational control (arrow, positive; stopped bar, negative). Question mark on Gcn2 refers to evidence suggesting that oxidative stress activates Gcn2 (Anda et al., 2017). Question mark on M-Stop refers to its possible involvement in regulation of rps/rpl translation. (B) and (C) Translational regulatory circuits discussed in this review. Left, original definition by (Alon, 2007), adapted from Molecular Biology of the Gene, 7^th edition. Right, translational regulatory motifs mediated by uORF-dependent control. In (B), Coherent FFL with AND node makes a persistent detector that only responds to a long-lived signal (Alon, 2007). However, Coherent FFL with OR node works differently, as described in the text.