Downloaded from rnajournal.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press eIF1 discriminates against suboptimal initiation sites to prevent excessive uORF translation genome-wide FUJUN ZHOU, HONGEN ZHANG, SHARDUL D. KULKARNI, JON R. LORSCH, and ALAN G. HINNEBUSCH Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Development, National Institutes of Health, Bethesda, Maryland 20892, USA ABSTRACT The translation preinitiation complex (PIC) scans the mRNA for an AUG codon in a favorable context. Previous findings sug- gest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and par- ticular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak con- text. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with the reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at certain NCCs initiating amino-terminal extensions, including those that direct mitochondrial localization of the GRS1 and ALA1 products, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself. Thus, eIF1 acts throughout the yeast translatome to discriminate against NCC start codons and AUGs in poor context; and impairing this function enhances the repressive effects of uORFs on CDS translation and alters the ratios of protein isoforms translated from near-cognate versus AUG start codons. Keywords: translation; initiation; accuracy; eIF1; yeast INTRODUCTION by GTPase activating protein eIF5, but Pi release is blocked by eIF1, whose presence also prevents more sta- Accurate identification of the translation initiation codon in ble binding of Met-tRNA Met in the “P ” state. Start-codon mRNA by ribosomes is crucial for the correct expression of i IN recognition triggers dissociation of eIF1 from the 40S sub- cellular proteins, and it generally occurs in eukaryotic cells unit, which in concert with other events allows P release by a scanning mechanism. The 43S preinitiation complex i from eIF2-GDP·P and accommodation of Met-tRNA Met (PIC), comprised of the small (40S) ribosomal subunit, i i in the P state of the 48S PIC (Supplemental Fig. S1). methionyl initiator tRNA (Met-tRNA Met) in a ternary com- IN i Subsequent dissociation of eIF2-GDP and other eIFs plex (TC) with GTP-bound eIF2, and several other initiation from the 48S PIC enables eIF5B-catalyzed subunit joining factors, attaches to the 5′ end of the mRNA and scans the and formation of an 80S initiation complex with Met- 5′UTR for an AUG start codon. In the scanning PIC, the TC tRNA Met base-paired to AUG in the P site, ready for the is bound in a relatively unstable state, dubbed “P ,” i OUT elongation phase of protein synthesis (for reviews, see suitable for inspecting successive triplets in the P decod- Hinnebusch 2014 and Hinnebusch 2017). ing site for complementarity with the anticodon of Met- Although eIF1 enhances rapid TC loading in the P tRNA Nucleotides surrounding the AUG, particularly at OUT i. conformation, it physically impedes rearrangement to P the −3 and +4 positions (relative to the AUG at +1 to +3) IN during scanning at non-AUG codons by clashing with —the “Kozak context”—also influence start codon recog- Met-tRNA (Rabl et al. 2011; Lomakin and Steitz 2013; nition. Hydrolysis of the GTP bound to eIF2 is stimulated i Hussain et al. 2014), thus requiring its displacement from the P site and subsequent dissociation from the 40S Corresponding authors: [email protected], [email protected] Article is online at http://www.rnajournal.org/cgi/doi/10.1261/rna. 073536.119. This is a work of the US Government. RNA (2020) 26:419–438; Published by Cold Spring Harbor Laboratory Press for the RNA Society 419 Downloaded from rnajournal.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press Zhou et al. subunit to allow start codon selection (Supplemental ened binding to the amino-terminal domain of eIF3c, the Fig. S1; Maag et al. 2005; Cheung et al. 2007). carboxy-terminal domain of eIF5, and the 40S subunit Consequently, mutations that weaken eIF1 binding to (Obayashi et al. 2017). the 40S subunit were found to increase initiation at the Although the role of yeast eIF1 in discriminating against near-cognate UUG triplet present at the third codon of near-cognate start codons or AUGs in poor context was HIS4 mRNA and thereby suppress the reduced translation well established for a small set of mRNAs, including conferred by substitution of the AUG start codon by the HIS4, SUI1, and various synthetic reporter mRNAs, its − his4-301 mutation, conferring the Sui (suppressor of initi- global importance in restricting usage of near-cognate ini- ation codon) phenotype. They also elevate initiation at tiation codons and AUG codons in the poor context in liv- UUG codons in synthetic reporter mRNAs, presumably ing yeast cells was unknown. Accordingly, here we have by increasing unscheduled dissociation of eIF1 from the used ribosome footprint profiling of the highly impaired − PIC in the absence of a perfect codon:anticodon helix in Sui variant eIF1-L96P to reveal the genome-wide function the P site (Martin-Marcos et al. 2011, 2013). Moreover, de- of eIF1 in promoting accurate start codon selection and creasing wild-type (WT) eIF1 abundance increases initia- proper expression of the yeast translatome. tion at near-cognates or AUGs in poor context, whereas overexpressing eIF1 suppresses these events (Valasek RESULTS et al. 2004; Alone et al. 2008; Ivanov et al. 2010; Saini et al. 2010; Martin-Marcos et al. 2011). The mechanistic eIF1-L96P decreases discrimination against near- link between eIF1 abundance and initiation accuracy is ex- cognate start codons genome-wide ploited to negatively autoregulate eIF1 expression, as the AUG codon of the eIF1 mRNA itself (SUI1 in yeast) is in sub- To determine whether impairing eIF1 function reduces dis- optimal context, and the frequency of its recognition is in- crimination against suboptimal initiation sites throughout versely related to eIF1 abundance (Ivanov et al. 2010; the yeast translatome, we conducted ribosomal footprint − − Martin-Marcos et al. 2011). The Sui mutations that weak- profiling of the strong Sui variant sui1-L96P in parallel en 40S binding by eIF1 also relax discrimination against with an isogenic WT strain. In addition to decreasing the the poor context of the SUI1 AUG codon and elevate accuracy of start site selection, L96P reduces the rate of ge- eIF1 expression, overcoming the autoregulation (Martin- neral translation initiation, decreasing the cell growth rate Marcos et al. 2011). Studies using a reconstituted mamma- (Martin-Marcos et al. 2011) and reducing the abundance lian initiation system indicate that eIF1 also discriminates and average size of bulk polysomes, to a greater extent against mRNAs containing very short 5′UTRs (Pestova than observed for mutations substituting conserved Lys and Kolupaeva 2002), also known to disfavor initiation in residues in direct contact with the 40S subunit, including yeast and mammalian cells (Hinnebusch 2011), possibly sui1-K56E and sui1-K60E (Supplemental Fig. S2A–E; in response to a lack of stabilizing mRNA nucleotides occu- Martin-Marcos et al. 2013). The L96P mutation also confers pying the mRNA exit channel of the 40S subunit. an ≈3.5-fold larger increase in the HIS4-lacZ UUG:AUG ini- The eIF1 mutation sui1-L96P was shown previously to tiation ratio relative to WT cells, versus compared to K56E suppress the reduced translation conferred by the AUG and K60E (Martin-Marcos et al. 2011). start codon mutation in his4-301 mRNA, conferring the Ribosome profiling entails deep-sequencing of mRNA − Sui phenotype, increasing by ∼18-fold the UUG:AUG ra- fragments protected from RNase digestion by translating tio of expression from two matched HIS4-lacZ reporters 80S ribosomes that were arrested on mRNA by treating differing only by an UUG or AUG start codon. In addition cell lysates with cycloheximide (Ribo-seq), conducted in to relaxing discrimination against UUG start codons at parallel with total mRNA sequencing (RNA-seq) (Ingolia HIS4, L96P conferred an ∼12-fold increase in expression et al. 2012). The TE of each mRNA is calculated for each of eIF1 itself, but a smaller, approximately fourfold in- strain as the ratio of total sequencing reads for ribosome- crease in SUI1 mRNA, suggesting an approximately three- protected fragments (RPFs) to total reads for mRNA frag- fold increase in translational efficiency (TE). The latter was ments mapping to the coding sequences (CDS) of each attributed primarily to relaxed discrimination against the gene; and is normalized to the average TE among all suboptimal Kozak context of the SUI1 AUG codon because mRNAs (which is assigned a value of one). Thus, TE values L96P had significantly less effect on eIF1 expression from a are expressed relative to the average TE in that experi- SUI1 mRNA altered to contain optimal Kozak context ment. Changes in TE in the L96P mutant versus WT (Martin-Marcos et al. 2011). Thus, the eIF1-L96P substitu- (ΔTEL96P) are expressed as the ratio TEL96P/TEWT, and sig- tion appears to diminish substantially eIF1 function in re- nificant changes were identified by DESeq analysis stricting initiation at both near-cognate (UUG) start (Anders and Huber 2010) of data from biological replicates codons and AUG codons in poor context.