Initiator tRNA Research Articles

Protein Synthesis Initiation in Eukaryotic Cells.

This review summarizes our current understanding of the major pathway for the initiation phase of protein synthesis in eukaryotic cells, with a focus on recent advances. We describe the major scanning or messenger RNA (mRNA) m7G cap-dependent mechanism, which is a highly coordinated and stepwise regulated process that requires the combined action of at least 12 distinct translation factors with initiator transfer RNA (tRNA), ribosomes, and mRNAs. We limit our review to studies involving either mammalian or budding yeast cells and factors, as these represent the two best-studied experimental systems, and only include a reference to other organisms where particular insight has been gained. We close with a brief description of what we feel are some of the major unknowns in eukaryotic initiation.

Translational Control and the Integrated Stress Response Regulate the Fate of UVB‐Irradiated Human Keratinocytes

Upon exposure to sublethal UVB irradiation, human keratinocytes transiently inhibit progression of the cell cycle to allow time for DNA repair and determination of cell fate. These processes are critical for evading the initiation of carcinogenesis in skin. Central to these adaptive processes is the Integrated stress response (ISR) that features repression of the initiation phase of protein synthesis by induced GCN2 phosphorylation of eIF2 (eIF2‐P). Phosphorylation of eIF2 prevents delivery of initiator tRNA to the ribosome machinery. Coincident with the reduction in global mRNA translation, eIF2‐P selectively enhances translation of mRNAs that participate in stress adaptation. In this study, we determined a cytoprotective mechanism for eIF2‐P in response to UVB in human keratinocytes. In response to UVB, genetically engineered loss of induced eIF2‐P lowered G1 arrest, DNA repair, and cellular senescence, coincident with enhanced apoptosis. Using polysome profiling and RNA‐seq analyses, we carried out genome‐wide measurements of translation in human keratinocytes. A collection of gene transcripts showed enhanced association with large polysomes in response to UVB, suggestive of enhanced translation efficiency. Network analysis of the preferentially translated genes indicated eIF2‐P regulation of the cell cycle, cell communication, stress responses, metabolism, and inflammation. One of the identified preferentially translated genes was CDKN1A (WAF1, CIP1) encoding p21 protein, that functions to block the progression from G1 to S phase of the cell cycle and to initiate cellular senescence. While enhanced levels of CDKN1A mRNA occurred in response to UVB independent of induced eIF2‐P, enhanced synthesis of p21 protein required the ISR. Two splicing variants of CDKN1A mRNA, V1 and V4, are expressed in keratinocytes, with V4 being preferentially translated following UVB induced eIF2‐P by a mechanism mediated in part by upstream ORFs situated in the 5′‐leader of CDKN1A mRNA. These results show that eIF2‐P is cytoprotective in response to UVB by a mechanism featuring translation of a specific splice variant of CDKN1A that facilitates G1 arrest and subsequent DNA repair.Support or Funding InformationNIH grants R01 GM049164 (RCW), F31 ES026517 (AEC), and R01 ES20866 (DFS)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Foldamers wave to the ribosome.

Ribosomes have now been shown to accept certain initiator tRNAs acylated with aromatic foldamer–dipeptides thereby enabling the translation of a peptide or protein with a short aromatic foldamer at the N-terminus. Some foldamer–peptide hybrids could be cyclized to generate macrocycles that present conformationally restricted peptide loops.

Serine Catabolism by SHMT2 Is Required for Proper Mitochondrial Translation Initiation and Maintenance of Formylmethionyl-tRNAs

Upon glucose restriction, eukaryotic cells upregulate oxidative metabolism to maintain homeostasis. Using genetic screens, we find that the mitochondrial serine hydroxymethyltransferase (SHMT2) is required for robust mitochondrial oxygen consumption and low glucose proliferation. SHMT2 catalyzes the first step in mitochondrial one-carbon metabolism, which, particularly in proliferating cells, produces tetrahydrofolate (THF)-conjugated one-carbon units used in cytoplasmic reactions despite the presence of a parallel cytoplasmic pathway. Impairing cytoplasmic one-carbon metabolism or blocking efflux of one-carbonunits from mitochondria does not phenocopy SHMT2 loss, indicating that a mitochondrial THF cofactor is responsible for the observed phenotype. The enzyme MTFMT utilizes one such cofactor, 10-formyl THF, producing formylmethionyl-tRNAs, specialized initiator tRNAs necessary for proper translation of mitochondrially encoded proteins. Accordingly, SHMT2 null cells specifically fail to maintain formylmethionyl-tRNA pools and mitochondrially encoded proteins, phenotypes similar to those observed in MTFMT-deficient patients. These findings provide a rationale for maintaining a compartmentalized one-carbon pathway in mitochondria.

Abstract LB-B28: Abietane natural products as novel therapeutics against solid tumors

Abstract Dysregulation of protein synthesis can lead to aberrant cellular proliferation and survival, a hallmark of cancer. Protein synthesis is a multistep process tightly controlled by specific translation factors. In eukaryotes, at the initiation point, the 43S preinitiation complex is formed by the association of several factors [eukaryotic initiation factor 2 (eIF2), GTP and methionyl initiator tRNA] with the 40S ribosomal subunit and the eukaryotic initiation factor 3 (eIF3). Over expression of eIFs has been reported in breast cancer, particularly in the most aggressive subtype, triple negative breast cancer (TNBC). More than one million global cases of breast cancer are diagnosed yearly and approximately 15% are characterized as triple negative breast cancer (TNBC), a subtype that lacks effective targeted therapy modalities. Recently, we generated a library of cell permeable abietane natural product derivatives (i.e. SJ6038) and showed that these compounds are more potent against TNBC over the estrogen receptor positive cellular models. These compounds were tagged with biotin for affinity enrichment and with a fluorescent chromophore to facilitate protein target(s) identification. A SILAC proteomics approach was used to distinguish specific binding targets from the non-specific ones. Gene Ontology analysis showed the targets identified were affiliated with regulation of protein synthesis. Ingenuity Pathway Analysis suggested that abietanes may exert their anticancer effects on critical biological pathways such as the eIF2, eIF4/p70S6K, and mTOR signaling pathways. Live cell experiments utilizing fluorescent abietane derivatives demonstrate that these probes localize to subsections of the ER, supporting the SILAC findings. Time of Exposure and Dose Response experiments further demonstrate that Abietane derivatives affect the eIF2 signaling axis. Using chemical biology and medicinal chemistry approaches, we show that abietane natural products preferentially promote cell death in TNBC cellular models by targeting the eIF2 signaling axis. These compounds will serve as unique chemical tools targeting the eIF2/eIF4 signaling axis and advance from hit- to lead for further preclinical development as a targeted therapy against aggressive solid tumors. Citation Format: Walter Lang, Taotao Ling, Jangsoon Lee, Naoto T. Ueno, Fatima Rivas. Abietane natural products as novel therapeutics against solid tumors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-B28.

Role of the Two Highly Conserved Features of Initiator tRNAs in Initiation of Proteins Synthesis in Eubacteria

Role of the Two Highly Conserved Features of Initiator tRNAs in Initiation of Proteins Synthesis in Eubacteria

Role of the Two Highly Conserved Features of Initiator tRNAs in the Initiation of Proteins and Synthesis in Eubacteria

Role of the Two Highly Conserved Features of Initiator tRNAs in the Initiation of Proteins and Synthesis in Eubacteria

Coevolution of the translational machinery optimizes initiation with unusual initiator tRNAs and initiation codons in mycoplasmas

ABSTRACTInitiator tRNAs (i-tRNAs) are characterized by the presence of three consecutive GC base pairs (GC/GC/GC) in their anticodon stems in all domains of life. However, many mycoplasmas possess unconventional i-tRNAs wherein the highly conserved sequence of GC/GC/GC is represented by AU/GC/GC, GC/GC/GU or AU/GC/GU. These mycoplasmas also tend to preferentially utilize non-AUG initiation codons. To investigate if initiation with the unconventional i-tRNAs and non-AUG codons in mycoplasmas correlated with the changes in the other components of the translation machinery, we carried out multiple sequence alignments of genes encoding initiation factors (IF), 16S rRNAs, and the ribosomal proteins such as uS9, uS12 and uS13. In addition, the occurrence of Shine-Dalgarno sequences in mRNAs was analyzed. We observed that in the mycoplasmas harboring AU/GC/GU i-tRNAs, a highly conserved position of R131 in IF3, is represented by P, F or Y and, the conserved C-terminal tail (SKR) of uS9 is represented by the TKR sequence. Using the Escherichia coli model, we show that the change of R131 in IF3 optimizes initiation with the AU/GC/GU i-tRNAs. Also, the SKR to TKR change in uS9 was compatible with the R131P variation in IF3 for initiation with the AU/GC/GU i-tRNA variant. Interestingly, the mycoplasmas harboring AU/GC/GU i-tRNAs are also human pathogens. We propose that these mycoplasmas might have evolved a relaxed translational apparatus to adapt to the environment they encounter in the host.

Highly chromophoric Cy5-methionine for N-terminal fluorescent tagging of proteins in eukaryotic translation systems

Despite significant advances on fluorescent labeling of target proteins to study their structural dynamics and function, there has been need for labeling with high quantum yield ensuring high sensitivity and selectivity without sacrificing the biological function of the protein. Here as a technical advancement over non-canonical amino acid incorporation, we provided a conceptual design of the N-terminal fluorescent tagging of proteins. Cy5-labeled methionine (Cy5-Met) was chemically synthesized, and then the purified Cy5-Met was coupled with synthetic human initiator tRNA by methionine tRNA synthetase. Cy5-Met-initiator tRNA (Cy5-Met-tRNAi) was purified and transfected into HeLa cells with HIV-Tat plasmid, resulting in an efficient production of Cy5-labeled HIV-Tat protein. Based on the universal requirement in translational initiation, the approach provides co-translational incorporation of N-terminal probe to a repertoire of proteins in the eukaryote system. This methodology has potential utility in the single molecule analysis of human proteins in vitro and in vivo for addressing to their complex biological structural and functional dynamics.

Fidelity of translation in the presence of mammalian mitochondrial initiation factor 3

Initiation factor 3 (IF3) is a conserved translation factor. Mutations in mitochondrial IF3 (IF3mt) have been implicated in disease pathology. Escherichia coli infCΔ55, compromised for IF3 activity, has provided an excellent heterologous system for IF3mt structure-function analysis. IF3mt allowed promiscuous initiation from AUA, AUU and ACG codons but avoided initiation with initiator tRNAs lacking the conserved 3GC pairs in their anticodon stems. Expression of IF3mt N-terminal domain, or IF3mt devoid of its typical N-, and C-terminal extensions improved fidelity of initiation in E. coli. The observations suggest that the IF3mt terminal extensions relax the fidelity of translational initiation in mitochondria.

Crystal Structure of the C-terminal Domain of Human eIF2D and Its Implications on Eukaryotic Translation Initiation

Protein synthesis is a key process in all living organisms. In eukaryotes, initiation factor 2 (eIF2) plays an important role in translation initiation as it selects and delivers the initiator tRNA to the small ribosomal subunit. Under stress conditions, phosphorylation of the α-subunit of eIF2 downregulates cellular protein synthesis. However, translation of certain mRNAs continues via the eIF2D-dependent non-canonical initiation pathway. The molecular mechanism of this process remains elusive. In addition, eIF2D plays a role in translation re-initiation and ribosome recycling. Currently, there has been no structural information of eIF2D. We have now determined the crystal structure of the C-terminal domains of eIF2D at 1.4-Å resolution. One domain has the fold similar to that of eIF1, which is crucial for the scanning and initiation codon selection. The second domain has a known SWIB/MDM2 fold, which was not observed before in other translation initiation factors. Our structure reveals atomic details of inter-domain interactions in the C-terminal part of eIF2D and sheds light on the possible role of these domains in eIF2D during translation.

Structural and Functional Insights into Human Re-initiation Complexes

After having translated short upstream open reading frames, ribosomes can re-initiate translation on the same mRNA. This process, referred to as re-initiation, controls the translation of a large fraction of mammalian cellular mRNAs, many of which are important in cancer. Key ribosomal binding proteins involved in re-initiation are the eukaryotic translation initiation factor 2D (eIF2D) or the homologous complex of MCT-1/DENR. We determined the structures of these factors bound to the human 40S ribosomal subunit in complex with initiator tRNA positioned on an mRNA start codon in the P-site using a combination of cryoelectron microscopy and X-ray crystallography. The structures, supported by biochemical experiments, reveal how eIF2D emulates the function of several canonical translation initiation factors by using three independent, flexibly connected RNA binding domains to simultaneously monitor codon-anticodon interactions in the ribosomal P-site and position the initiator tRNA.

Methionyl-tRNA synthetase overexpression is associated with poor clinical outcomes in non-small cell lung cancer

BackgroundMethionyl-tRNA synthetase (MRS) plays a critical role in initiating translation by transferring Met to the initiator tRNA (tRNAiMet) and protection against ROS-mediated damage, suggesting that its overexpression is related to cancer growth and drug resistance. In this study, the clinical implication of MRS expression in non-small cell lung cancer (NSCLC) was evaluated.MethodsImmunoblot and immunohistochemical (IHC) analyses were performed using tissue lysates and formalin-fixed paraffin embedded (FFPE) tissue blocks from wild type C57BL/6, LSL-Kras G12D, and LSL-Kras G12D:p53fl/fl mice. For human studies, 12 paired adjacent normal appearing lung tissue lysates and cancer tissue lysates, in addition to 231 FFPE tissue samples, were used.ResultsMRS was weakly expressed in the spleen and intestinal epithelium and only marginally expressed in the kidney, liver, and lungs of wild type C57BL/6 mice. On the other hand, MRS was strongly expressed in the neoplastic region of lung tissue from LSL-Kras G12D and LSL-Kras G12D:p53fl/fl mice. Immunoblot analysis of the human normal appearing adjacent and lung cancer paired tissue lysates revealed cancer-specific MRS overexpression, which was related to mTORC1 activity. IHC analysis of the 231 FFPE lung cancer tissue samples showed that MRS expression was frequently detected in the cytoplasm of lung cancer cells (179 out of 231, 77.4%), with a small proportion (73 out of 231, 31.6%) also showing nuclear expression. The proportion of cases with positive MRS expression was higher in the advanced pStage subgroup (P = 0.018, χ2-test) and cases with MRS expression also had shorter DFS (161.6 vs 142.3, P = 0.014, log-rank test).ConclusionsTaken together, MRS is frequently overexpressed in NSCLC. Moreover, MRS is related to mTORC1 activity and its overexpression is associated with poor clinical outcomes, indicating that it has potential as a putative therapeutic target.

Coupling of Translation Initiation and Termination Does Not Depend on the Mode of Initiation.

Recently we described a novel phenomenon observed during eukaryotic translation in a cell-free system: the coupling of initiation and termination on different mRNA molecules. Here we show that the phenomenon does not depend on a special mode of initiation. The mRNAs with certain leader sequences known to require different determinants for successful initiation were examined. Even in a case of using the intergenic internal ribosome entry site (IRES) of cricket paralysis virus RNA as the leader sequence, while no initiation factors are required, the effect of coupling is well expressed, including trials in the presence of hippuristanol as an inhibitor of eIF4A. Thus, the effect persists in the absence of scanning and does not depend on initiator tRNA and eIF2. The results suggest that the initiation factors are not involved in the coupling mechanism.

Contributions of the N- and C-Terminal Domains of Initiation Factor 3 to Its Functions in the Fidelity of Initiation and Antiassociation of the Ribosomal Subunits.

Initiation factor 3 (IF3) is one of the three conserved prokaryotic translation initiation factors essential for protein synthesis and cellular survival. Bacterial IF3 is composed of a conserved architecture of globular N- and C-terminal domains (NTD and CTD) joined by a linker region. IF3 is a ribosome antiassociation factor which also modulates selection of start codon and initiator tRNA. All the functions of IF3 have been attributed to its CTD by in vitro studies. However, the in vivo relevance of these findings has not been investigated. By generating complete and partial IF3 (infC) knockouts in Escherichia coli and by complementation analyses using various deletion constructs, we show that while the CTD is essential for E. coli survival, the NTD is not. Polysome profiles reaffirm that CTD alone can bind to the 30S ribosomal subunit and carry out the ribosome antiassociation function. Importantly, in the absence of the NTD, bacterial growth is compromised, indicating a role for the NTD in the fitness of cellular growth. Using reporter assays for in vivo initiation, we show that the NTD plays a crucial role in the fidelity function of IF3 by avoiding (i) initiation from non-AUG codons and (ii) initiation by initiator tRNAs lacking the three highly conserved consecutive GC pairs (in the anticodon stem) known to function in concert with IF3.IMPORTANCE Initiation factor 3 regulates the fidelity of eubacterial translation initiation by ensuring the formation of an initiation complex with an mRNA bearing a canonical start codon and with an initiator tRNA at the ribosomal P site. Additionally, IF3 prevents premature association of the 50S ribosomal subunit with the 30S preinitiation complex. The significance of our work in Escherichia coli is in demonstrating that while the C-terminal domain alone sustains E. coli for its growth, the N-terminal domain adds to the fidelity of initiation of protein synthesis and to the fitness of the bacterial growth.

AtaT blocks translation initiation by N-acetylation of the initiator tRNAfMet.

Toxin-antitoxin (TA) loci are prevalent in bacterial genomes. They are suggested to play a central role in dormancy and persister states. Under normal growth conditions, TA toxins are neutralized by their cognate antitoxins, and under stress conditions, toxins are freed and inhibit essential cellular processes using a variety of mechanisms. Here we characterize ataR-ataT, a novel TA system, from enterohemorrhagic Escherichia coli. We show that the toxin AtaT is a GNAT family enzyme that transfers an acetyl group from acetyl coenzyme A to the amine group of the methionyl aminoacyl moiety of initiator tRNA. AtaT specifically modifies Met-tRNAfMet, but no other aminoacyl-tRNAs, including the elongator Met-tRNAMet. We demonstrate that once acetylated, AcMet-tRNAfMet fails to interact with initiation factor-2 (IF2), resulting in disruption of the translation initiation complex. This work reveals a new mechanism of translation inhibition and confirms Met-tRNAfMet as a prime target to efficiently block cell growth.

Eukaryotic aspects of translation initiation brought into focus.

In all organisms, mRNA-directed protein synthesis is catalysed by ribosomes. Although the basic aspects of translation are preserved in all kingdoms of life, important differences are found in the process of translation initiation, which is rate-limiting and the most important step for translation regulation. While great strides had been taken towards a complete structural understanding of the initiation of translation in eubacteria, our understanding of the eukaryotic process, which includes numerous eukaryotic-specific initiation factors, was until recently limited owing to a lack of structural information. In this review, we discuss recent results in the field that provide an increasingly complete molecular description of the eukaryotic initiation process. The structural snapshots obtained using a range of methods now provide insights into the architecture of the initiation complex, start-codon recognition by the initiator tRNA and the process of subunit joining. Future advances will require both higher-resolution insights into previously characterized complexes and mapping of initiation factors that control translation on an additional level by interacting only peripherally or transiently with ribosomal subunits.This article is part of the themed issue 'Perspectives on the ribosome'.

The structure of an E. coli tRNAfMet A1-U72 variant shows an unusual conformation of the A1-U72 base pair.

Translation initiation in eukaryotes and archaea involves a methionylated initiator tRNA delivered to the ribosome in a ternary complex with e/aIF2 and GTP. Eukaryotic and archaeal initiator tRNAs contain a highly conserved A1–U72 base pair at the top of the acceptor stem. The importance of this base pair to discriminate initiator tRNAs from elongator tRNAs has been established previously using genetics and biochemistry. However, no structural data illustrating how the A1–U72 base pair participates in the accurate selection of the initiator tRNAs by the translation initiation systems are available. Here, we describe the crystal structure of a mutant E. coli initiator tRNAfMetA1–U72, aminoacylated with methionine, in which the C1:A72 mismatch at the end of the tRNA acceptor stem has been changed to an A1–U72 base pair. Sequence alignments show that the mutant E. coli tRNA is a good mimic of archaeal initiator tRNAs. The crystal structure, determined at 2.8 Å resolution, shows that the A1–U72 pair adopts an unusual arrangement. A1 is in a syn conformation and forms a single H-bond interaction with U72. This interaction requires protonation of the N1 atom of A1. Moreover, the 5′ phosphoryl group folds back into the major groove of the acceptor stem and interacts with the N7 atom of G2. A possible role of this unusual geometry of the A1–U72 pair in the recognition of the initiator tRNA by its partners during eukaryotic and archaeal translation initiation is discussed.

Mitochondrial malfunction in vanishing white matter disease: a disease of the cytosolic translation machinery.

Mitochondrial malfunction in vanishing white matter disease: a disease of the cytosolic translation machinery.

Structure of a 30S pre-initiation complex stalled by GE81112 reveals structural parallels in bacterial and eukaryotic protein synthesis initiation pathways.

In bacteria, the start site and the reading frame of the messenger RNA are selected by the small ribosomal subunit (30S) when the start codon, typically an AUG, is decoded in the P-site by the initiator tRNA in a process guided and controlled by three initiation factors. This process can be efficiently inhibited by GE81112, a natural tetrapeptide antibiotic that is highly specific toward bacteria. Here GE81112 was used to stabilize the 30S pre-initiation complex and obtain its structure by cryo-electron microscopy. The results obtained reveal the occurrence of changes in both the ribosome conformation and initiator tRNA position that may play a critical role in controlling translational fidelity. Furthermore, the structure highlights similarities with the early steps of initiation in eukaryotes suggesting that shared structural features guide initiation in all kingdoms of life.