Components Of Translational Machinery Research Articles

In Vitro Studies to Define the Cell-Surface and Intracellular Targets of Polyarginine-Conjugated Sodium Borocaptate as a Potential Delivery Agent for Boron Neutron Capture Therapy.

Boron neutron capture therapy (BNCT) requires pharmaceutical innovations and molecular-based evidence of effectiveness to become a standard cancer therapeutic in the future. Recently, in Japan, 4-borono-L-phenylalanine (BPA) was approved as a boron agent for BNCT against head and neck (H&N) cancers. H&N cancer appears to be a suitable target for BPA-BNCT, because the expression levels of L-type amino acid transporter 1 (LAT1), one of the amino acid transporters responsible for BPA uptake, are elevated in most cases of H&N cancer. However, in other types of cancer including malignant brain tumors, LAT1 is not always highly expressed. To expand the possibility of BNCT for these cases, we previously developed poly-arginine peptide (polyR)-conjugated mercaptoundecahydrododecaborate (BSH). PolyR confers the cell membrane permeability and tumor selectivity of BSH. However, the molecular determinants for the properties are not fully understood. In this present study, we have identified the cluster of differentiation 44 (CD44) protein and translational machinery proteins as a major cell surface target and intracellular targets of BSH-polyR, respectively. CD44, also known as a stem cell-associated maker in various types of cancer, is required for the cellular uptake of polyR-conjugated molecules. We showed that BSH-polyR was predominantly delivered to a CD44High cell population of cancer cells. Once delivered, BSH-polyR interacted with the translational machinery components, including the initiation factors, termination factors, and poly(A)-biding protein (PABP). As a proof of principle, we performed BSH-polyR-based BNCT against glioma stem-like cells and revealed that BSH-polyR successfully induced BNCT-dependent cell death specifically in CD44High cells. Bioinformatics analysis indicated that BSH-polyR would be suitable for certain types of malignant tumors. Our results shed light on the biochemical properties of BSH-polyR, which may further contribute to the therapeutic optimization of BSH-BNCT in the future.

Measuring mRNA translation in neuronal processes and somata by tRNA-FRET.

In neurons, the specific spatial and temporal localization of protein synthesis is of great importance for function and survival. Here, we visualized tRNA and protein synthesis events in fixed and live mouse primary cortical culture using fluorescently-labeled tRNAs. We were able to characterize the distribution and transport of tRNAs in different neuronal sub-compartments and to study their association with the ribosome. We found that tRNA mobility in neural processes is lower than in somata and corresponds to patterns of slow transport mechanisms, and that larger tRNA puncta co-localize with translational machinery components and are likely the functional fraction. Furthermore, chemical induction of long-term potentiation (LTP) in culture revealed up-regulation of mRNA translation with a similar effect in dendrites and somata, which appeared to be GluR-dependent 6 h post-activation. Importantly, measurement of protein synthesis in neurons with high resolutions offers new insights into neuronal function in health and disease states.

Coordinated Regulation of Rsd and RMF for Simultaneous Hibernation of Transcription Apparatus and Translation Machinery in Stationary-Phase Escherichia coli.

Transcription and translation in growing phase of Escherichia coli, the best-studied model prokaryote, are coupled and regulated in coordinate fashion. Accordingly, the growth rate-dependent control of the synthesis of RNA polymerase (RNAP) core enzyme (the core component of transcription apparatus) and ribosomes (the core component of translation machinery) is tightly coordinated to keep the relative level of transcription apparatus and translation machinery constant for effective and efficient utilization of resources and energy. Upon entry into the stationary phase, transcription apparatus is modulated by replacing RNAP core-associated sigma (promoter recognition subunit) from growth-related RpoD to stationary-phase-specific RpoS. The anti-sigma factor Rsd participates for the efficient replacement of sigma, and the unused RpoD is stored silent as Rsd–RpoD complex. On the other hand, functional 70S ribosome is transformed into inactive 100S dimer by two regulators, ribosome modulation factor (RMF) and hibernation promoting factor (HPF). In this review article, we overview how we found these factors and what we know about the molecular mechanisms for silencing transcription apparatus and translation machinery by these factors. In addition, we provide our recent findings of promoter-specific transcription factor (PS-TF) screening of the transcription factors involved in regulation of the rsd and rmf genes. Results altogether indicate the coordinated regulation of Rsd and RMF for simultaneous hibernation of transcription apparatus and translation machinery.

Bias for 3'-Dominant Codon Directional Asymmetry in Theoretical Minimal RNA Rings.

Aminoacyl tRNA synthetases ligate tRNAs specifically with their cognate amino acid. These synthetases are among life's earliest proteins, class II tRNA synthetases (cognates A, D, F, G, H, K, N, P, S, and T) presumably preceding class I tRNA synthetases (cognates C, E, I, L, M, Q, R, V, W, and Y). Classification of codons into palindromic (structure XYX), 5'-dominant (YXX), and 3'-dominant (XXY) (Codon Directional Asymmetry [CDA]) shows that class II tRNA synthetases aminoacylate amino acids associated with XXY. Our working hypothesis expects bias for XXY codons in primordial RNAs, such as theoretical minimal RNA rings, designed in silico to mimic life's earliest RNAs. Twenty-five RNA rings have been computed, which code over a minimal length (22 nucleotides) for a start codon, stop codon, and one and only one codon for each of the 20 amino acids, and form stem-loop hairpins preventing degradation; these 25 minimal RNAs are the only ones matching these constraints and they seem homologous to consensus tRNA sequences. This similarity defined candidate RNA ring anticodons and corresponding cognate amino acids. Here, analyses of RNA ring codon contents confirm bias for XXY codons in 13 among 14 RNA rings with unequal XXY and YXX codon numbers. This bias increases with the genetic code integration order of the RNA ring's cognate amino acid across and within tRNA synthetase classes, suggesting that evolutionary processes, and not physicochemical constraints, produced the association between CDA and tRNA synthetase classes. The self-referential hypothesis for genetic code origin, a very complete genetic code evolutionary hypothesis integrating many translational machinery components, predicts best among genetic code evolutionary hypotheses CDA biases in RNA rings. The RNA rings' simple design inadvertently reproduces CDAs predicted by the genetic code's structure, confirming theoretical minimal RNA rings as good proxies for life's earliest RNAs.

Numerous cultivated and uncultivated viruses encode ribosomal proteins

Viruses modulate ecosystems by directly altering host metabolisms through auxiliary metabolic genes. However, viral genomes are not known to encode the core components of translation machinery, such as ribosomal proteins (RPs). Here, using reference genomes and global-scale viral metagenomic datasets, we identify 14 different RPs across viral genomes arising from cultivated viral isolates and metagenome-assembled viruses. Viruses tend to encode dynamic RPs, easily exchangeable between ribosomes, suggesting these proteins can replace cellular versions in host ribosomes. Functional assays confirm that the two most common virus-encoded RPs, bS21 and bL12, are incorporated into 70S ribosomes when expressed in Escherichia coli. Ecological distribution of virus-encoded RPs suggests some level of ecosystem adaptations as aquatic viruses and viruses of animal-associated bacteria are enriched for different subsets of RPs. Finally, RP genes are under purifying selection and thus likely retained an important function after being horizontally transferred into virus genomes.

Evolutionary insights into Trm112-methyltransferase holoenzymes involved in translation between archaea and eukaryotes.

Protein synthesis is a complex and highly coordinated process requiring many different protein factors as well as various types of nucleic acids. All translation machinery components require multiple maturation events to be functional. These include post-transcriptional and post-translational modification steps and methylations are the most frequent among these events. In eukaryotes, Trm112, a small protein (COG2835) conserved in all three domains of life, interacts and activates four methyltransferases (Bud23, Trm9, Trm11 and Mtq2) that target different components of the translation machinery (rRNA, tRNAs, release factors). To clarify the function of Trm112 in archaea, we have characterized functionally and structurally its interaction network using Haloferax volcanii as model system. This led us to unravel that methyltransferases are also privileged Trm112 partners in archaea and that this Trm112 network is much more complex than anticipated from eukaryotic studies. Interestingly, among the identified enzymes, some are functionally orthologous to eukaryotic Trm112 partners, emphasizing again the similarity between eukaryotic and archaeal translation machineries. Other partners display some similarities with bacterial methyltransferases, suggesting that Trm112 is a general partner for methyltransferases in all living organisms.

Abstract 4137: Inhibition of protein synthesis with polyamine depletion in high-risk neuroblastoma

Abstract The MYC family of oncogenes coordinately deregulate cellular programs that link cell cycle progression with the requisite biomass and energy creation. Neuroblastoma is a MYC-driven, commonly lethal pediatric tumor, with a high proportion of tumors containing MYCN amplification. Polyamine synthesis is a pathway downstream of MYC, with polyamines functioning, in part, to support protein synthesis. ODC1 encodes ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, and we found that ODC1 is co-amplified with MYCN in a subset of high-risk neuroblastomas (~8%). Difluoromethylornithine (DFMO) is an irreversible inhibitor or ornithine decarboxylase and is an approved agent for the treatment of Trypanosomiasis. We have previously shown that DFMO inhibits tumor progression and synergizes with chemotherapy in murine mouse models of neuroblastoma, and we postulate that inhibition of protein synthesis is a predominant mechanism of anti-tumor activity. The effects of polyamine depletion on specific components of protein translational machinery have yet to be defined. Here, we show inhibition of global protein synthesis of up to 90% after treatment with various concentrations of DFMO in vitro, with a larger decrease in protein synthesis generally correlating with the presence of MYCN amplification. We found that colony formation is inhibited at DFMO exposures as low as 150 to 500 μM DFMO, which are achievable concentrations in adults based on previous clinical trials. We also show via isoelectric focusing that treatment of neuroblastoma cells in vitro with DFMO leads to a marked decrease in the proportion of hypusinated, or active, eIF5A (an elongation factor important in translation of polyproline stretches and possibly broader influence on translation initiation). To interrogate influences of polyamine depletion on the cap-dependent translation eIF4F complex, we evaluated for changes in the phosphorylation of 4E-BP1 in DFMO treatment conditions, though no changes in phosphorylation were seen compared to controls. Studies to further elucidate the protein synthetic components affected by polyamine depletion are ongoing, as are studies to assess for selective effects of DFMO on polyproline-containing proteins via eIF5A. Further elucidation of the mechanisms of DFMO activity will identify opportunities for drug synergy and provide a responder hypothesis to test in upcoming pivotal Phase 2 and 3 clinical trial of DFMO in the treatment of neuroblastoma. Citation Format: Andrea T. Flynn, Kangning Liu, Annette Vu, Michael D. Hogarty. Inhibition of protein synthesis with polyamine depletion in high-risk neuroblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4137.

Mutations in Gene fusA1 as a Novel Mechanism of Aminoglycoside Resistance in Clinical Strains of Pseudomonas aeruginosa.

Resistance of clinical strains of Pseudomonas aeruginosa to aminoglycosides can result from production of transferable aminoglycoside-modifying enzymes, of 16S rRNA methylases, and/or mutational derepression of intrinsic multidrug efflux pump MexXY(OprM). We report here the characterization of a new type of mutant that is 4- to 8-fold more resistant to 2-deoxystreptamine derivatives (e.g., gentamicin, amikacin, and tobramycin) than the wild-type strain PAO1. The genetic alterations of three in vitro mutants were mapped on fusA1 and found to result in single amino acid substitutions in domains II, III, and V of elongation factor G (EF-G1A), a key component of translational machinery. Transfer of the mutated fusA1 alleles into PAO1 reproduced the resistance phenotype. Interestingly, fusA1 mutants with other amino acid changes in domains G, IV, and V of EF-G1A were identified among clinical strains with decreased susceptibility to aminoglycosides. Allelic-exchange experiments confirmed the relevance of these latter mutations and of three other previously reported alterations located in domains G and IV. Pump MexXY(OprM) partly contributed to the resistance conferred by the mutated EF-G1A variants and had additive effects on aminoglycoside MICs when mutationally upregulated. Altogether, our data demonstrate that cystic fibrosis (CF) and non-CF strains of P. aeruginosa can acquire a therapeutically significant resistance to important aminoglycosides via a new mechanism involving mutations in elongation factor EF-G1A.

Interactions between the Translation Machinery and Microtubules.

Microtubules are components of eukaryotic cytoskeleton that are involved in the transport of various components from the nucleus to the cell periphery and back. They also act as a platform for assembly of complex molecular ensembles. Ribonucleoprotein (RNP) complexes, such as ribosomes and mRNPs, are transported over significant distances (e.g. to neuronal processes) along microtubules. The association of RNPs with microtubules and their transport along these structures are essential for compartmentalization of protein biosynthesis in cells. Microtubules greatly facilitate assembly of stress RNP granules formed by accumulation of translation machinery components during cell stress response. Microtubules are necessary for the cytoplasm-to-nucleus transport of proteins, including ribosomal proteins. At the same time, ribosomal proteins and RNA-binding proteins can influence cell mobility and cytoplasm organization by regulating microtubule dynamics. The molecular mechanisms underlying the association between the translation machinery components and microtubules have not been studied systematically; the results of such studies are mostly fragmentary. In this review, we attempt to fill this gap by summarizing and discussing the data on protein and RNA components of the translation machinery that directly interact with microtubules or microtubule motor proteins.

Epitranscriptomic m6A Regulation of Axon Regeneration in the Adult Mammalian Nervous System

N6-methyladenosine (m6A) affects multiple aspects of mRNA metabolism and regulates developmental transitions by promoting mRNA decay. Little is known about the role of m6A in the adult mammalian nervous system. Here we report that sciatic nerve lesion elevates levels of m6A-tagged transcripts encoding many regeneration-associated genes and protein translation machinery components in the adult mouse dorsal root ganglion (DRG). Single-base resolution m6A-CLIP mapping further reveals a dynamic m6A landscape in the adult DRG upon injury. Loss of either m6A methyltransferase complex component Mettl14 or m6A-binding protein Ythdf1 globally attenuates injury-induced protein translation in adult DRGs and reduces functional axon regeneration in the peripheral nervous system invivo. Furthermore, Pten deletion-induced axon regeneration of retinal ganglion neurons in the adult central nervous system is attenuated upon Mettl14 knockdown. Our study reveals a critical epitranscriptomic mechanism in promoting injury-induced protein synthesis and axon regeneration in the adult mammalian nervous system.

Strategies of biochemical adaptation for hibernation in a South American marsupial, Dromiciops gliroides: 2. Control of the Akt pathway and protein translation machinery.

When faced with harsh environmental conditions, the South American marsupial, monito del monte (Dromiciops gliroides), reduces its body temperature and uses either daily torpor or multiday hibernation to survive. This study used ELISA and multiplex assays to characterize the responses to hibernation by three regulatory components of protein translation machinery [p-eIF2α(S51), p-eIF4E(S209), p-4EBP(Thr37/46)] and eight targets involved in upstream signaling control of translation [p-IGF-1R(Tyr1135/1136), PTEN(S380), p-Akt(S473), p-GSK-3α(S21), p-GSK-3β(S9), p-TSC2(S939), p-mTOR(S2448), and p70S6K(T412)]. Liver, brain and kidney were analyzed comparing control and hibernation (4days continuous torpor) conditions. In the liver, increased phosphorylation of IGF-1R, Akt, GSK-3β, TSC2, mTOR, eIF2α, and 4EBP (1.60-1.98 fold compared to control) occurred during torpor suggesting that the regulatory phosphorylation cascade and protein synthesis remained active during torpor. However, responses by brain and kidney differed; torpor resulted in increased phosphorylation of GSK-3β (2.15-4.17 fold) and TSC2 (2.03-3.65 fold), but phosphorylated Akt decreased (to 34-62% of control levels). Torpor also led to an increase in phosphorylated eIF2α (1.4 fold) content in the brain. These patterns of differential protein phosphorylation in brain and kidney were indicative of suppression of protein translation but also could suggest an increase in antioxidant and anti-apoptotic signaling during torpor. Previous studies of liver metabolism in hibernating eutherian mammals have shown that Akt kinase and its downstream signaling components play roles in facilitating hypometabolism by suppressing energy expensive anabolic processes during torpor. However, the results in this study reveal differences between eutherian and marsupial hibernators, suggesting alternative actions of liver Akt during torpor.

Viral internal ribosomal entry sites: four classes for one goal.

To ensure efficient propagation, viruses need to rapidly produce viral proteins after cell entrance. Since viral genomes do not encode any components of the protein biosynthesis machinery, viral proteins must be produced by the host cell. To hi-jack the host cellular translation, viruses use a great variety of distinct strategies. Many single-stranded positive-sensed RNA viruses contain so-called internal ribosome entry sites (IRESs). IRESs are structural RNA motifs that have evolved to specific folds that recruit the host ribosomes on the viral coding sequences in order to synthesize viral proteins. In host canonical translation, recruitment of the translation machinery components is essentially guided by the 5' cap (m7 G) of mRNA. In contrast, IRESs are able to promote efficient ribosome assembly internally and in cap-independent manner. IRESs have been categorized into four classes, based on their length, nucleotide sequence, secondary and tertiary structures, as well as their mode of action. Classes I and II require the assistance of cellular auxiliary factors, the eukaryotic intiation factors (eIF), for efficient ribosome assembly. Class III IRESs require only a subset of eIFs whereas Class IV, which are the more compact, can promote translation without any eIFs. Extensive functional and structural investigations of IRESs over the past decades have allowed a better understanding of their mode of action for viral translation. Because viral translation has a pivotal role in the infectious program, IRESs are therefore attractive targets for therapeutic purposes. WIREs RNA 2018, 9:e1458. doi: 10.1002/wrna.1458 This article is categorized under: Translation > Ribosome Structure/Function Translation > Translation Mechanisms RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes.

Proteomic analysis of polyribosomes identifies splicing factors as potential regulators of translation during mitosis.

Precise regulation of mRNA translation is critical for proper cell division, but little is known about the factors that mediate it. To identify mRNA-binding proteins that regulate translation during mitosis, we analyzed the composition of polysomes from interphase and mitotic cells using unbiased quantitative mass-spectrometry (LC–MS/MS). We found that mitotic polysomes are enriched with a subset of proteins involved in RNA processing, including alternative splicing and RNA export. To demonstrate that these may indeed be regulators of translation, we focused on heterogeneous nuclear ribonucleoprotein C (hnRNP C) as a test case and confirmed that it is recruited to elongating ribosomes during mitosis. Then, using a combination of pulsed SILAC, metabolic labeling and ribosome profiling, we showed that knockdown of hnRNP C affects both global and transcript-specific translation rates and found that hnRNP C is specifically important for translation of mRNAs that encode ribosomal proteins and translation factors. Taken together, our results demonstrate how proteomic analysis of polysomes can provide insight into translation regulation under various cellular conditions of interest and suggest that hnRNP C facilitates production of translation machinery components during mitosis to provide daughter cells with the ability to efficiently synthesize proteins as they enter G1 phase.

Mutant eIF2B leads to impaired mitochondrial oxidative phosphorylation in vanishing white matter disease.

Eukaryotic translation initiation factor 2B (eIF2B) is a master regulator of protein synthesis under normal and stress conditions. Mutations in any of the five genes encoding its subunits lead to vanishing white matter (VWM) disease, a recessive genetic deadly illness caused by progressive loss of white matter in the brain. In this study we used fibroblasts, which are not involved in the disease, to demonstrate the involvement of eIF2B in mitochondrial function and abundance. Mass spectrometry of total proteome of mouse embryonic fibroblasts (MEFs) isolated from Eif2b5R132H/R132H mice revealed unbalanced stoichiometry of proteins involved in oxidative phosphorylation and of mitochondrial translation machinery components, among others. Mutant MEFs exhibit 55% decrease in oxygen consumption rate per mtDNA content and 47% increase in mitochondrial abundance (p<0.005), reflecting adaptation to energy requirements. A more robust eIF2B-associated oxidative respiration deficiency was found in mutant primary astrocytes, which exhibit >3-fold lower ATP-linked respiration per cell despite a 2-fold increase in mtDNA content (p<0.03). The 2-fold increase in basal and stimulated glycolysis in mutant astrocytes (p≤0.03), but not in MEFs, demonstrates their higher energetic needs and further explicates their involvement in the disease. The data demonstrate the critical role of eIF2B in tight coordination of expression from nuclear and mitochondrial genomes and illuminates the importance of mitochondrial function in VWM pathology. Further dissection of the signaling network associated with eIF2B function will help generating therapeutic strategies for VWM disease and possibly other neurodegenerative disorders.

Translation system engineering in Escherichia coli enhances non-canonical amino acid incorporation into proteins.

The ability to site-specifically incorporate non-canonical amino acids (ncAAs) into proteins has made possible the study of protein structure and function in fundamentally new ways, as well as the bio synthesis of unnatural polymers. However, the task of site-specifically incorporating multiple ncAAs into proteins with high purity and yield continues to present a challenge. At the heart of this challenge lies the lower efficiency of engineered orthogonal translation system components compared to their natural counterparts (e.g., translation elements that specifically use a ncAA and do not interact with the cell's natural translation apparatus). Here, we show that evolving and tuning expression levels of multiple components of an engineered translation system together as a whole enhances ncAA incorporation efficiency. Specifically, we increase protein yield when incorporating multiple p-azido-phenylalanine(pAzF) residues into proteins by (i) evolving the Methanocaldococcus jannaschii p-azido-phenylalanyl-tRNA synthetase anti-codon binding domain, (ii) evolving the elongation factor Tu amino acid-binding pocket, and (iii) tuning the expression of evolved translation machinery components in a single vector. Use of the evolved translation machinery in a genomically recoded organism lacking release factor one enabled enhanced multi-site ncAA incorporation into proteins. We anticipate that our approach to orthogonal translation system development will accelerate and expand our ability to site-specifically incorporate multiple ncAAs into proteins and biopolymers, advancing new horizons for synthetic and chemical biotechnology. Biotechnol. Bioeng. 2017;114: 1074-1086. © 2016 Wiley Periodicals, Inc.

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.

Minimum-noise production of translation factor eIF4G maps to a mechanistically determined optimal rate control window for protein synthesis.

Gene expression noise influences organism evolution and fitness. The mechanisms determining the relationship between stochasticity and the functional role of translation machinery components are critical to viability. eIF4G is an essential translation factor that exerts strong control over protein synthesis. We observe an asymmetric, approximately bell-shaped, relationship between the average intracellular abundance of eIF4G and rates of cell population growth and global mRNA translation, with peak rates occurring at normal physiological abundance. This relationship fits a computational model in which eIF4G is at the core of a multi-component–complex assembly pathway. This model also correctly predicts a plateau-like response of translation to super-physiological increases in abundance of the other cap-complex factors, eIF4E and eIF4A. Engineered changes in eIF4G abundance amplify noise, demonstrating that minimum stochasticity coincides with physiological abundance of this factor. Noise is not increased when eIF4E is overproduced. Plasmid-mediated synthesis of eIF4G imposes increased global gene expression stochasticity and reduced viability because the intrinsic noise for this factor influences total cellular gene noise. The naturally evolved eIF4G gene expression noise minimum maps within the optimal activity zone dictated by eIF4G's mechanistic role. Rate control and noise are therefore interdependent and have co-evolved to share an optimal physiological abundance point.

5'-Terminal nucleotide variations in human cytoplasmic tRNAHisGUG and its 5'-halves.

Transfer RNAs (tRNAs) are fundamental adapter components of translational machinery. tRNAs can further serve as a source of tRNA-derived noncoding RNAs that play important roles in various biological processes beyond translation. Among all species of tRNAs, tRNAHisGUG has been known to uniquely contain an additional guanosine residue at the −1 position (G−1) of its 5′-end. To analyze this −1 nucleotide in detail, we developed a TaqMan qRT-PCR method that can distinctively quantify human mature cytoplasmic tRNAHisGUG containing G−1, U−1, A−1, or C−1 or lacking the −1 nucleotide (starting from G1). Application of this method to the mature tRNA fraction of BT-474 breast cancer cells revealed the presence of tRNAHisGUG containing U−1 as well as the one containing G−1. Moreover, tRNA lacking the −1 nucleotide was also detected, thus indicating the heterogeneous expression of 5′-tRNAHisGUG variants. A sequence library of sex hormone-induced 5′-tRNA halves (5′-SHOT-RNAs), identified via cP-RNA-seq of a BT-474 small RNA fraction, also demonstrated the expression of 5′-tRNAHisGUG halves containing G−1, U−1, or G1 as 5′-terminal nucleotides. Although the detected 5′-nucleotide species were identical, the relative abundances differed widely between mature tRNA and 5′-half from the same BT-474 cells. The majority of mature tRNAs contained the −1 nucleotide, whereas the majority of 5′-halves lacked this nucleotide, which was biochemically confirmed using a primer extension assay. These results reveal the novel identities of tRNAHisGUG molecules and provide insights into tRNAHisGUG maturation and the regulation of tRNA half production.

In Vivo Biosynthesis of a β-Amino Acid-Containing Protein.

It has recently been reported that ribosomes from erythromycin-resistant Escherichia coli strains, when isolated in S30 extracts and incubated with chemically mis-acylated tRNA, can incorporate certain β-amino acids into full length DHFR in vitro. Here we report that wild-type E. coli EF-Tu and phenylalanyl-tRNA synthetase collaborate with these mutant ribosomes and others to incorporate β(3)-Phe analogs into full length DHFR in vivo. E. coli harboring the most active mutant ribosomes are robust, with a doubling time only 14% longer than wild-type. These results reveal the unexpected tolerance of E. coli and its translation machinery to the β(3)-amino acid backbone and should embolden in vivo selections for orthogonal translational machinery components that incorporate diverse β-amino acids into proteins and peptides. E. coli harboring mutant ribosomes may possess the capacity to incorporate many non-natural, non-α-amino acids into proteins and other sequence-programmed polymeric materials.

TRN-A-RS Acts As Biomarker for Cancer and Other Diseases

Background: tRNA and ARS, tRN-A-RS, pivot the translational machinery. How their aberrances correlate to diseases, neurological conditions, metabolic disorders, and cancer is the purpose of this study. Methods: Information was retrieved from the literature and from databases, such as OMIM and MITOMAP, related to disease and cancer. Changes of expressions of ARSs were assessed from analyzing NGS data available from TCGA, ENCODE and lately from roadmap epigenomics. Results: A total of 647 tRNAs and 37 ARSs reside in the genomic pool of human, aberrant expressions of some of the nuclear tRNAs and cytoplasmic ARSs correlate predominately to cancer, while anomalous pathways in mitochondria relate more to other diseases. tRNA fragments juggle between tumor suppressor and oncogenic pathways. Brain cancer and neurological disorders seem to gain impetus from translation machinery components. However, investigation of the causes of the diseases particularly cancer due to the aberrations continues to be hamstrung by the lack of deep sequencing reads of tRNAs in different cell lines, and this needs to be addressed. Conclusions: Increased cell proliferation requires elevated protein synthesis levels and makes the correlation between tumour cells and deregulated tRN-A-RS components in translation plausible. We draw the network of tRN-A-RS with cancer and other diseases, and present and expand on some of the hypotheses on the underlying molecular mechanisms, opening up new avenues for research.