Scanning Preinitiation Complex Research Articles

Pervasive downstream RNA hairpins dynamically dictate start-codon selection

Translational reprogramming allows organisms to adapt to changing conditions. Upstream start codons (uAUGs), which are prevalently present in mRNAs, have crucial roles in regulating translation by providing alternative translation start sites1–4. However, what determines this selective initiation of translation between conditions remains unclear. Here, by integrating transcriptome-wide translational and structural analyses during pattern-triggered immunity in Arabidopsis, we found that transcripts with immune-induced translation are enriched with upstream open reading frames (uORFs). Without infection, these uORFs are selectively translated owing to hairpins immediately downstream of uAUGs, presumably by slowing and engaging the scanning preinitiation complex. Modelling using deep learning provides unbiased support for these recognizable double-stranded RNA structures downstream of uAUGs (which we term uAUG-ds) being responsible for the selective translation of uAUGs, and allows the prediction and rational design of translating uAUG-ds. We found that uAUG-ds-mediated regulation can be generalized to human cells. Moreover, uAUG-ds-mediated start-codon selection is dynamically regulated. After immune challenge in plants, induced RNA helicases that are homologous to Ded1p in yeast and DDX3X in humans resolve these structures, allowing ribosomes to bypass uAUGs to translate downstream defence proteins. This study shows that mRNA structures dynamically regulate start-codon selection. The prevalence of this RNA structural feature and the conservation of RNA helicases across kingdoms suggest that mRNA structural remodelling is a general feature of translational reprogramming.

IRQC, a surveillance pathway for 40S ribosomal quality control during mRNA translation initiation.

Post-translational modification of ribosomal proteins enables rapid and dynamic regulation of protein biogenesis. Site-specific ubiquitylation of 40S ribosomal proteins uS10 and eS10 plays a key role during ribosome-associated quality control (RQC). Distinct, and previously functionally ambiguous, ubiquitylation events on the 40S proteins uS3 and uS5 are induced by diverse proteostasis stressors that impact translation activity. Here, we identify the ubiquitin ligase RNF10 and the deubiquitylating enzyme USP10 as the key enzymes that regulate uS3 and uS5 ubiquitylation. Prolonged uS3 and uS5 ubiquitylation results in 40S, but not 60S, ribosomal protein degradation in a manner independent of canonical autophagy. We show that blocking progression of either scanning or elongating ribosomes past the start codon triggers site-specific ubiquitylation events on ribosomal proteins uS5 and uS3. This study identifies and characterizes a distinct arm in the RQC pathway, initiation RQC (iRQC), that acts on 40S ribosomes during translation initiation to modulate translation activity and capacity.

Ribosome queuing enables non-AUG translation to be resistant to multiple protein synthesis inhibitors.

Aberrant translation initiation at non-AUG start codons is associated with multiple cancers and neurodegenerative diseases. Nevertheless, how non-AUG translation may be regulated differently from canonical translation is poorly understood. Here, we used start codon-specific reporters and ribosome profiling to characterize how translation from non-AUG start codons responds to protein synthesis inhibitors in human cells. These analyses surprisingly revealed that translation of multiple non-AUG-encoded reporters and the endogenous GUG-encoded DAP5 (eIF4G2/p97) mRNA is resistant to cycloheximide (CHX), a translation inhibitor that severely slows but does not completely abrogate elongation. Our data suggest that slowly elongating ribosomes can lead to queuing/stacking of scanning preinitiation complexes (PICs), preferentially enhancing recognition of weak non-AUG start codons. Consistent with this model, limiting PIC formation or scanning sensitizes non-AUG translation to CHX. We further found that non-AUG translation is resistant to other inhibitors that target ribosomes within the coding sequence but not those targeting newly initiated ribosomes. Together, these data indicate that ribosome queuing enables mRNAs with poor initiation context-namely, those with non-AUG start codons-to be resistant to pharmacological translation inhibitors at concentrations that robustly inhibit global translation.

EIF1 Loop 2 interactions with Met-tRNAi control the accuracy of start codon selection by the scanning preinitiation complex.

The eukaryotic 43S preinitiation complex (PIC), bearing initiator methionyl transfer RNA (Met-tRNAi) in a ternary complex (TC) with eukaryotic initiation factor 2 (eIF2)-GTP, scans the mRNA leader for an AUG codon in favorable context. AUG recognition evokes rearrangement from an open PIC conformation with TC in a "POUT" state to a closed conformation with TC more tightly bound in a "PIN" state. eIF1 binds to the 40S subunit and exerts a dual role of enhancing TC binding to the open PIC conformation while antagonizing the PIN state, necessitating eIF1 dissociation for start codon selection. Structures of reconstituted PICs reveal juxtaposition of eIF1 Loop 2 with the Met-tRNAi D loop in the PIN state and predict a distortion of Loop 2 from its conformation in the open complex to avoid a clash with Met-tRNAi We show that Ala substitutions in Loop 2 increase initiation at both near-cognate UUG codons and AUG codons in poor context. Consistently, the D71A-M74A double substitution stabilizes TC binding to 48S PICs reconstituted with mRNA harboring a UUG start codon, without affecting eIF1 affinity for 40S subunits. Relatively stronger effects were conferred by arginine substitutions; and no Loop 2 substitutions perturbed the rate of TC loading on scanning 40S subunits in vivo. Thus, Loop 2-D loop interactions specifically impede Met-tRNAi accommodation in the PIN state without influencing the POUT mode of TC binding; and Arg substitutions convert the Loop 2-tRNAi clash to an electrostatic attraction that stabilizes PIN and enhances selection of poor start codons in vivo.

Translation initiation of alphavirus mRNA reveals new insights into the topology of the 48S initiation complex.

The topology and dynamics of the scanning ribosomal 43S pre-initiation complex (PIC) bound to mRNA and initiation factors (eIFs) are probably the least understood aspects of translation initiation in eukaryotes. Recently, we described a trapping mechanism in alphavirus that stalls the PIC during scanning of viral mRNA. Using this model, we were able to snapshot for the first time the eIF4A helicase bound to mRNA in a 48S initiation complex assembled in vitro. This interaction was only detected in the presence of the natural stem loop structure (DLP) located downstream from the AUG in viral mRNA that promoted stalling of the PIC, suggesting that DLP stability was enough to jam the helicase activity of eIF4A in a fraction of assembled 48S complexes. However, a substantial proportion of DLP mRNA molecules were effectively unwound by eIF4A in vitro, an activity that alphaviruses counteract in infected cells by excluding eIF4A from viral factories. Our data indicated that eIF4A–mRNA contact occurred in (or near) the ES6S region of the 40S subunit, suggesting that incoming mRNA sequences penetrate through the ES6S region during the scanning process. We propose a topological model of the scanning PIC and how some viruses have exploited this topology to translate their mRNAs with fewer eIF requirements.

Molecular Landscape of the Ribosome Pre-initiation Complex during mRNA Scanning: Structural Role for eIF3c and Its Control by eIF5

During eukaryotic translation initiation, eIF3 binds the solvent-accessible side of the 40S ribosome and recruits the gate-keeper protein eIF1 and eIF5 to the decoding center. This is largely mediated by the N-terminal domain (NTD) of eIF3c, which can be divided into three parts: 3c0, 3c1, and 3c2. The N-terminal part, 3c0, binds eIF5 strongly but only weakly to the ribosome-binding surface of eIF1, whereas 3c1 and 3c2 form a stoichiometric complex with eIF1. 3c1 contacts eIF1 through Arg-53 and Leu-96, while 3c2 faces 40S protein uS15/S13, to anchor eIF1 to the scanning pre-initiation complex (PIC). We propose that the 3c0:eIF1 interaction diminishes eIF1 binding to the 40S, whereas 3c0:eIF5 interaction stabilizes the scanning PIC by precluding this inhibitory interaction. Upon start codon recognition, interactions involving eIF5, and ultimately 3c0:eIF1 association, facilitate eIF1 release. Our results reveal intricate molecular interactions within the PIC, programmed for rapid scanning-arrest at the start codon.

The Scanning Mechanism of Eukaryotic Translation Initiation

In eukaryotes, the translation initiation codon is generally identified by the scanning mechanism, wherein every triplet in the messenger RNA leader is inspected for complementarity to the anticodon of methionyl initiator transfer RNA (Met-tRNAi). Binding of Met-tRNAi to the small (40S) ribosomal subunit, in a ternary complex (TC) with eIF2-GTP, is stimulated by eukaryotic initiation factor 1 (eIF1), eIF1A, eIF3, and eIF5, and the resulting preinitiation complex (PIC) joins the 5' end of mRNA preactivated by eIF4F and poly(A)-binding protein. RNA helicases remove secondary structures that impede ribosome attachment and subsequent scanning. Hydrolysis of eIF2-bound GTP is stimulated by eIF5 in the scanning PIC, but completion of the reaction is impeded at non-AUG triplets. Although eIF1 and eIF1A promote scanning, eIF1 and possibly the C-terminal tail of eIF1A must be displaced from the P decoding site to permit base-pairing between Met-tRNAi and the AUG codon, as well as to allow subsequent phosphate release from eIF2-GDP. A second GTPase, eIF5B, catalyzes the joining of the 60S subunit to produce an 80S initiation complex that is competent for elongation.

The Interaction between Eukaryotic Initiation Factor 1A and eIF5 Retains eIF1 within Scanning Preinitiation Complexes

Scanning of the mRNA transcript by the preinitiation complex (PIC) requires a panel of eukaryotic initiation factors, which includes eIF1 and eIF1A, the main transducers of stringent AUG selection. eIF1A plays an important role in start codon recognition; however, its molecular contacts with eIF5 are unknown. Using nuclear magnetic resonance, we unveil eIF1A's binding surface on the carboxyl-terminal domain of eIF5 (eIF5-CTD). We validated this interaction by observing that eIF1A does not bind to an eIF5-CTD mutant, altering the revealed eIF1A interaction site. We also found that the interaction between eIF1A and eIF5-CTD is conserved between humans and yeast. Using glutathione S-transferase pull-down assays of purified proteins, we showed that the N-terminal tail (NTT) of eIF1A mediates the interaction with eIF5-CTD and eIF1. Genetic evidence indicates that overexpressing eIF1 or eIF5 suppresses the slow growth phenotype of eIF1A-NTT mutants. These results suggest that the eIF1A-eIF5-CTD interaction during scanning PICs contributes to the maintenance of eIF1 within the open PIC.

β-Hairpin Loop of Eukaryotic Initiation Factor 1 (eIF1) Mediates 40 S Ribosome Binding to Regulate Initiator tRNAMet Recruitment and Accuracy of AUG Selection in Vivo

Recognition of the translation initiation codon is thought to require dissociation of eIF1 from the 40 S ribosomal subunit, enabling irreversible GTP hydrolysis (Pi release) by the eIF2·GTP·Met-tRNAi ternary complex (TC), rearrangement of the 40 S subunit to a closed conformation incompatible with scanning, and stable binding of Met-tRNAi to the P site. The crystal structure of a Tetrahymena 40 S·eIF1 complex revealed several basic amino acids in eIF1 contacting 18 S rRNA, and we tested the prediction that their counterparts in yeast eIF1 are required to prevent premature eIF1 dissociation from scanning ribosomes at non-AUG triplets. Supporting this idea, substituting Lys-60 in helix α1, or either Lys-37 or Arg-33 in β-hairpin loop-1, impairs binding of yeast eIF1 to 40 S·eIF1A complexes in vitro, and it confers increased initiation at UUG codons (Sui(-) phenotype) or lethality, in a manner suppressed by overexpressing the mutant proteins or by an eIF1A mutation (17-21) known to impede eIF1 dissociation in vitro. The eIF1 Sui(-) mutations also derepress translation of GCN4 mRNA, indicating impaired ternary complex loading, and this Gcd(-) phenotype is likewise suppressed by eIF1 overexpression or the 17-21 mutation. These findings indicate that direct contacts of eIF1 with 18 S rRNA seen in the Tetrahymena 40 S·eIF1 complex are crucial in yeast to stabilize the open conformation of the 40 S subunit and are required for rapid TC loading and ribosomal scanning and to impede rearrangement to the closed complex at non-AUG codons. Finally, we implicate the unstructured N-terminal tail of eIF1 in blocking rearrangement to the closed conformation in the scanning preinitiation complex.

Coordinated Movements of Eukaryotic Translation Initiation Factors eIF1, eIF1A, and eIF5 Trigger Phosphate Release from eIF2 in Response to Start Codon Recognition by the Ribosomal Preinitiation Complex*

Accurate recognition of the start codon in an mRNA by the eukaryotic translation preinitiation complex (PIC) is essential for proper gene expression. The process is mediated by eukaryotic translation initiation factors (eIFs) in conjunction with the 40 S ribosomal subunit and (initiator) tRNA(i). Here, we provide evidence that the C-terminal tail (CTT) of eIF1A, which we previously implicated in start codon recognition, moves closer to the N-terminal domain of eIF5 when the PIC encounters an AUG codon. Importantly, this movement is coupled to dissociation of eIF1 from the PIC, a critical event in start codon recognition, and is dependent on the scanning enhancer elements in the eIF1A CTT. The data further indicate that eIF1 dissociation must be accompanied by the movement of the eIF1A CTT toward eIF5 in order to trigger release of phosphate from eIF2, which converts the latter to its GDP-bound state. Our results also suggest that release of eIF1 from the PIC and movement of the CTT of eIF1A are triggered by the same event, most likely accommodation of tRNA(i) in the P site of the 40 S subunit driven by base pairing between the start codon in the mRNA and the anticodon in tRNA(i). Finally, we show that the C-terminal domain of eIF5 is responsible for the factor's activity in antagonizing eIF1 binding to the PIC. Together, our data provide a more complete picture of the chain of molecular events that is triggered when the scanning PIC encounters an AUG start codon in the mRNA.

The Indispensable N-Terminal Half of eIF3j/HCR1 Cooperates with its Structurally Conserved Binding Partner eIF3b/PRT1-RRM and with eIF1A in Stringent AUG Selection

Despite recent progress in our understanding of the numerous functions of individual subunits of eukaryotic translation initiation factor (eIF) 3, little is known on the molecular level. Using NMR spectroscopy, we determined the first solution structure of an interaction between eIF3 subunits. We revealed that a conserved tryptophan residue in the human eIF3j N-terminal acidic motif (NTA) is held in the helix α1 and loop 5 hydrophobic pocket of the human eIF3b RNA recognition motif (RRM). Mutating the corresponding “pocket” residues in its yeast orthologue reduces cellular growth rate, eliminates eIF3j/HCR1 association with eIF3b/PRT1 in vitro and in vivo, affects 40S occupancy of eIF3, and produces a leaky scanning defect indicative of a deregulation of the AUG selection process. Unexpectedly, we found that the N-terminal half of eIF3j/HCR1 containing the NTA is indispensable and sufficient for wild-type growth of yeast cells. Furthermore, we demonstrate that deletion of either j/HCR1 or its N-terminal half only, or mutation of the key tryptophan residues results in the severe leaky scanning phenotype partially suppressible by overexpressed eIF1A, which is thought to stabilize properly formed preinitiation complexes at the correct start codon. These findings indicate that eIF3j/HCR1 remains associated with the scanning preinitiation complexes and does not dissociate from the small ribosomal subunit upon mRNA recruitment, as previously believed. Finally, we provide further support for earlier mapping of the ribosomal binding site for human eIF3j by identifying specific interactions of eIF3j/HCR1 with small ribosomal proteins RPS2 and RPS23 located in the vicinity of the mRNA entry channel. Taken together, we propose that eIF3j/HCR1 closely cooperates with the eIF3b/PRT1 RRM and eIF1A on the ribosome to ensure proper formation of the scanning-arrested conformation required for stringent AUG recognition.

Ribosomal Protein L33 Is Required for Ribosome Biogenesis, Subunit Joining, and Repression of GCN4 Translation

We identified a mutation in the 60S ribosomal protein L33A (rpl33a-G76R) that elicits derepression of GCN4 translation (Gcd- phenotype) by allowing scanning preinitiation complexes to bypass inhibitory upstream open reading frame 4 (uORF4) independently of prior uORF1 translation and reinitiation. At 37 degrees C, rpl33a-G76R confers defects in 60S biogenesis comparable to those produced by the deletion of RPL33A (DeltaA). At 28 degrees C, however, the 60S biogenesis defect is less severe in rpl33a-G76R than in DeltaA cells, yet rpl33a-G76R confers greater derepression of GCN4 and a larger reduction in general translation. Hence, it appears that rpl33a-G76R has a stronger effect on ribosomal-subunit joining than does a comparable reduction of wild-type 60S levels conferred by DeltaA. We suggest that rpl33a-G76R alters the 60S subunit in a way that impedes ribosomal-subunit joining and thereby allows 48S rRNA complexes to abort initiation at uORF4, resume scanning, and initiate downstream at GCN4. Because overexpressing tRNAiMet suppresses the Gcd- phenotype of rpl33a-G76R cells, dissociation of tRNAiMet from the 40S subunit may be responsible for abortive initiation at uORF4 in this mutant. We further demonstrate that rpl33a-G76R impairs the efficient processing of 35S and 27S pre-rRNAs and reduces the accumulation of all four mature rRNAs, indicating an important role for L33 in the biogenesis of both ribosomal subunits.

Tetracycline-aptamer-mediated translational regulation in yeast.

We describe post-transcriptional gene regulation in yeast based on direct RNA-ligand interaction. Tetracycline-dependent translational regulation could be imposed via specific aptamers inserted at two different positions in the 5' untranslated region (5'UTR). Translation in vivo was suppressed up to ninefold upon addition of tetracycline. Repression via an aptamer located near the start codon (cap-distal) in the 5'UTR was more effective than repression via a cap-proximal position. On the other hand, suppression in a cell-free system reached maximally 50-fold and was most effective via a cap-proximal aptamer. Examination of the kinetics of tetracycline-dependent translational inhibition in vitro revealed that preincubation of tetracycline and mRNA before starting translation led not only to the fastest onset of inhibition but also the most effective repression. The differences between the behaviour of the regulatory system in vivo and in vitro are likely to be related to distinct properties of mRNP structure and mRNA accessibility in intact cells as opposed to cell-extracts. Tetracycline-dependent regulation was also observed after insertion of an uORF sequence upstream of the aptamer, indicating that our system also targets reinitiating ribosomes. Polysomal gradient analyses provided insight into the mechanism of regulation. Cap-proximal insertion inhibits binding of the 43S complex to the cap structure whereas start-codon-proximal aptamers interfere with formation of the 80S ribosome, probably by blocking the scanning preinitiation complex.

Regulation and function of non-AUG-initiated proto-oncogenes

A small, yet growing, number of cellular eukaryotic mRNAs encoding important regulatory proteins, such as c-myc and other proto-oncogenes, initiate translation from a non-AUG codon, usually in addition to initiating at a downstream AUG. The efficiency of non-AUG initiation on these natural cellular mRNAs varies considerably and appears to be governed by several features, including the codon sequence, the context surrounding the codon and the secondary structure of the transcript. In addition to factors which control the overall efficiency of c-myc non-AUG initiation, the relative efficiency of the upstream non-AUG initiation compared with the AUG initiation changes during the growth of cells. As lymphoid and fibroblast cells approach high densities in culture there is a sustained 5–10-fold induction in the synthesis of the non-AUG-initiated c-Myc 1 protein to levels comparable to or greater than the AUG-initiated c-Myc 2 protein. This increased efficiency of c-myc non-AUG initiation, due to methionine depletion of the growth medium, suggests that the scanning preinitiation complex can be regulated to enhance the recognition of a suboptimal non-AUG codon. The significance of non-AUG initiation for the growth-regulatory genes is illustrated by the different localizations of the int-2, bFGF and hck non-AUG-initiated proteins, the disruption of the c-myc and lyl-1 non-AUG initiation in tumor-derived cell lines, and the distinct biological function of the non-AUG-initiated forms of bFGF.