Queuosine‐modified tRNAs confer nutritional control of protein translation

Global protein translation as well as translation at the codon level can be regulated by tRNA modifications. In eukaryotes, levels of tRNA queuosinylation reflect the bioavailability of the precursor queuine, which is salvaged from the diet and gut microbiota. We show here that nutritionally determined Q‐tRNA levels promote Dnmt2‐mediated methylation of tRNA Asp and control translational speed of Q‐decoded codons as well as at near‐cognate codons. Deregulation of translation upon queuine depletion results in unfolded proteins that trigger endoplasmic reticulum stress and activation of the unfolded protein response, both in cultured human cell lines and in germ‐free mice fed with a queuosine‐deficient diet. Taken together, our findings comprehensively resolve the role of this anticodon tRNA modification in the context of native protein translation and describe a novel mechanism that links nutritionally determined modification levels to effective polypeptide synthesis and cellular homeostasis.     

A role for tRNA modifications in genome structure and codon usage

Transfer RNA (tRNA) gene content is a differentiating feature of genomes that contributes to the efficiency of the translational apparatus, but the principles shaping tRNA gene copy number and codon composition are poorly understood. Here, we report that the emergence of two specific tRNA modifications shaped the structure and composition of all extant genomes. Through the analysis of more than 500 genomes, we identify two kingdom-specific tRNA modifications as major contributors that separated archaeal, bacterial, and eukaryal genomes in terms of their tRNA gene composition. We show that, contrary to prior observations, genomic codon usage and tRNA gene frequencies correlate in all kingdoms if these two modifications are taken into account and that presence or absence of these modifications explains patterns of gene expression observed in previous studies. Finally, we experimentally demonstrate that human gene expression levels correlate well with genomic codon composition if these identified modifications are considered.     

The Arabidopsis mitochondrial membrane‐bound ubiquitin protease UBP27 contributes to mitochondrial morphogenesis

Mitochondria are essential organelles with dynamic morphology and function. Post‐translational modifications (PTMs), which include protein ubiquitination, are critically involved in animal and yeast mitochondrial dynamics. How PTMs contribute to plant mitochondrial dynamics is just beginning to be elucidated, and mitochondrial enzymes involved in ubiquitination have not been reported from plants. In this study, we identified an Arabidopsis mitochondrial localized ubiquitin protease, UBP27, through a screen that combined bioinformatics and fluorescent fusion protein targeting analysis. We characterized UBP27 with respect to its membrane topology and enzymatic activities, and analysed the mitochondrial morphological changes in UBP27T‐DNA insertion mutants and overexpression lines. We have shown that UBP27 is embedded in the mitochondrial outer membrane with an N_in–C_out orientation and possesses ubiquitin protease activities in vitro. UBP27 demonstrates similar sub‐cellular localization, domain structure, membrane topology and enzymatic activities with two mitochondrial deubiquitinases, yeast ScUBP16 and human HsUSP30, which indicated that these proteins are functional orthologues in eukaryotes. Although loss‐of‐function mutants of UBP27 do not show obvious phenotypes in plant growth and mitochondrial morphology, UBP27 overexpression can change mitochondrial morphology from rod to spherical shape and reduce the mitochondrial association of dynamin‐related protein 3 (DRP3) proteins, large GTPases that serve as the main mitochondrial fission factors. Thus, our study has uncovered a plant ubiquitin protease that plays a role in mitochondrial morphogenesis possibly through modulation of the function of organelle division proteins.     

High‐yield secretion of recombinant proteins from the microalga Chlamydomonas reinhardtii

Microalga‐based biomanufacturing of recombinant proteins is attracting growing attention due to its advantages in safety, metabolic diversity, scalability and sustainability. Secretion of recombinant proteins can accelerate the use of microalgal platforms by allowing post‐translational modifications and easy recovery of products from the culture media. However, currently, the yields of secreted recombinant proteins are low, which hampers the commercial application of this strategy. This study aimed at expanding the genetic tools for enhancing secretion of recombinant proteins in Chlamydomonas reinhardtii, a widely used green microalga as a model organism and a potential industrial biotechnology platform. We demonstrated that the putative signal sequence from C. reinhardtii gametolysin can assist the secretion of the yellow fluorescent protein Venus into the culture media. To increase the secretion yields, Venus was C‐terminally fused with synthetic glycomodules comprised of tandem serine (Ser) and proline (Pro) repeats of 10 and 20 units [hereafter (SP)_n, wherein n = 10 or 20]. The yields of the (SP)_n‐fused Venus were higher than Venus without the glycomodule by up to 12‐fold, with the maximum yield of 15 mg/L. Moreover, the presence of the glycomodules conferred an enhanced proteolytic protein stability. The Venus‐(SP)_n proteins were shown to be glycosylated, and a treatment of the cells with brefeldin A led to a suggestion that glycosylation of the (SP)_n glycomodules starts in the endoplasmic reticulum (ER). Taken together, the results demonstrate the utility of the gametolysin signal sequence and (SP)_n glycomodule to promote a more efficient biomanufacturing of microalgae‐based recombinant proteins.     

Dissecting the contributions of GC content and codon usage to gene expression in the model alga Chlamydomonas reinhardtii

The efficiency of gene expression in all organisms depends on the nucleotide composition of the coding region. GC content and codon usage are the two key sequence features known to influence gene expression, but the underlying molecular mechanisms are not entirely clear. Here we have determined the relative contributions of GC content and codon usage to the efficiency of nuclear gene expression in the unicellular green alga Chlamydomonas reinhardtii. By comparing gene variants that encode an identical amino acid sequence but differ in their GC content and/or codon usage, we show that codon usage is the key factor determining translational efficiency and, surprisingly, also mRNA stability. By contrast, unfavorable GC content affects gene expression at the level of the chromatin structure by triggering heterochromatinization. We further show that mutant algal strains that permit high‐level transgene expression are less susceptible to epigenetic transgene suppression and do not establish a repressive chromatin structure at the transgenic locus. Our data disentangle the relationship between GC content and codon usage, and suggest simple strategies to overcome the transgene expression problem in Chlamydomonas.     

Phosphorylation of Argonaute proteins affects mRNA binding and is essential for microRNA‐guided gene silencing in vivo

Argonaute proteins associate with microRNAs and are key components of gene silencing pathways. With such a pivotal role, these proteins represent ideal targets for regulatory post‐translational modifications. Using quantitative mass spectrometry, we find that a C‐terminal serine/threonine cluster is phosphorylated at five different residues in human and Caenorhabditis elegans. In human, hyper‐phosphorylation does not affect microRNA binding, localization, or cleavage activity of Ago2. However, mRNA binding is strongly affected. Strikingly, on Ago2 mutants that cannot bind microRNAs or mRNAs, the cluster remains unphosphorylated indicating a role at late stages of gene silencing. In C. elegans, the phosphorylation of the conserved cluster of ALG‐1 is essential for microRNA function in vivo. Furthermore, a single point mutation within the cluster is sufficient to phenocopy the loss of its complete phosphorylation. Interestingly, this mutant retains its capacity to produce and bind microRNAs and represses expression when artificially tethered to an mRNA. Altogether, our data suggest that the phosphorylation state of the serine/threonine cluster is important for Argonaute–mRNA interactions.     

Misannotations of the genes encoding sugar N‐formyltransferases

Tens of thousands of bacterial genome sequences are now known due to the development of rapid and inexpensive sequencing technologies. An important key in utilizing these vast amounts of data in a biologically meaningful way is to infer the function of the proteins encoded in the genomes via bioinformatics techniques. Whereas these approaches are absolutely critical to the annotation of gene function, there are still issues of misidentifications, which must be experimentally corrected. For example, many of the bacterial DNA sequences encoding sugar N‐formyltransferases have been annotated as l ‐methionyl‐tRNA transferases in the databases. These mistakes may be due in part to the fact that until recently the structures and functions of these enzymes were not well known. Herein we describe the misannotation of two genes, WP_088211966.1 and WP_096244125.1, from Shewanella spp. and Pseudomonas congelans, respectively. Although the proteins encoded by these genes were originally suggested to function as l ‐methionyl‐tRNA transferases, we demonstrate that they actually catalyze the conversion of dTDP‐4‐amino‐4,6‐dideoxy‐d ‐glucose to dTDP‐4‐formamido‐4,6‐dideoxy‐d ‐glucose utilizing N¹⁰‐formyltetrahydrofolate as the carbon source. For this analysis, the genes encoding these enzymes were cloned and the corresponding proteins purified. X‐ray structures of the two proteins were determined to high resolution and kinetic analyses were conducted. Both enzymes display classical Michaelis–Menten kinetics and adopt the characteristic three‐dimensional structural fold previously observed for other sugar N‐formyltransferases. The results presented herein will aid in the future annotation of these fascinating enzymes.     

Efficient tRNA degradation and quantification in Escherichia coli cell extract using RNase‐coated magnetic beads: A key step toward codon emancipation

Emancipating sense codons toward a minimized genetic code is of significant interest to science and engineering. A key approach toward sense codon emancipation is the targeted in vitro removal of native tRNA. However, challenges remain such as the insufficient depletion of tRNA in lysate‐based in vitro systems and the high cost of the purified components system (PURE). Here we used RNase‐coated superparamagnetic beads to efficiently degrade E. coli endogenous tRNA. The presented method removes >99% of tRNA in cell lysates, while partially preserving cell‐free protein synthesis activity. The resulting tRNA‐depleted lysate is compatible with in vitro‐transcribed synthetic tRNA for the production of peptides and proteins. Additionally, we directly measured residual tRNA using quantitative real‐time PCR. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 33:1401–1407, 2017     

Functional interactions in vivo between suppressor tRNA and mutationally altered ribosomal protein S4

Summary Ribosomal mutants (rpsD) which are associated with a generally increased translational ambiguity were investigated for their effects in vivo on individual tRNA species using suppressor tRNAs as models. It was found that nonsense suppression is either increased, unaffected or decreased depending on the codon context and the rpsD allele involved as well as the nature of the suppressor tRNA. Missense suppression of AGA and AGG by glyT(SuAGA/G) tRNA as well as UGG by glyT(SuUGG-8) tRNA is unaffected whereas suppression of UGG by glyT(SuUGA/G) or glyV(SuUGA/G) tRNA is decreased in the presence of an rpsD mutation. The effects on suppressor tRNA are thus not correlated with the ribosomal ambiguity (Ram) phenotype of the rpsD mutants used in this study. It is suggested that the mutationally altered ribosomes are changed in functional interactions with the suppressor tRNA itself rather than with the competing translational release factor(s) or cognate aminoacyl tRNA. The structure of suppressor tRNA, particularly the anticodon loop, and the suppressed codon as well as the codon context determine the allele specific functional interactions with these ribosomal mutations.     

Identification of TTA codon containing genes in <Emphasis Type="Italic">Frankia</Emphasis> and exploration of the role of tRNA in regulating these genes

The TTA codon, one of the six available codons for the amino acid leucine, is the rarest codon among the high GC genomes of Actinobacteria including Frankia. This codon has been implicated in various regulatory mechanisms involving secondary metabolism and morphological development. TTA-mediated gene regulation is well documented in Streptomyces coelicolor, but that role has not been investigated in other Actinobacteria including Frankia. Among the various Actinomycetes with a GC content of more than 70%, Frankia genomes had the highest percentages of TTA-containing genes ranging from 5.2 to 10.68% of the genome. In contrast, TTA-bearing genes comprised 1.7, 3.4 and 4.1% of the Streptomyces coelicolor, S. avermitilis and Nocardia farcinia genomes, respectively. We analyzed their functional role, evolutionary significance, horizontal acquisition and the codon-anticodon interaction. The TTA-bearing genes were found to be well represented in metabolic genes involved in amino acid transport and secondary metabolism. A reciprocal Blast search reveal that many of the TTA-bearing genes have orthologs in the other Frankia genomes, and some of these orthologous genes also have a TTA codon in them. The gene expression level of TTA-containing genes was estimated by the use of the codon adaption index (CAI), and the CAI values were found to have a positive correlation with the GC3 (GC content at the 3rd codon position). A full-atomic 3D model of the leucine tRNA recognizing the TTA (UUA) codon was generated and utilized for in silico docking to determine binding affinity in codon-anticodon interaction. We found a proficient codon-anticodon interaction for this codon which is perhaps why so many genes hold on to this rare codon without compromising their translational efficiency.