The polyphyletic origins of glycyl-tRNA synthetase and lysyl-tRNA synthetase and their implications.

I analyzed the polyphyletic origin of glycyl-tRNA synthetase (GlyRS) and lysyl-tRNA synthetase (LysRS), making plausible the following implications. The fact that the genetic code needed to evolve aminoacyl-tRNA synthetases (ARSs) only very late would be in perfect agreement with a late origin, in the main phyletic lineages, of both GlyRS and LysRS. Indeed, as suggested by the coevolution theory, since the genetic code was structured by biosynthetic relationships between amino acids and as these occurred on tRNA-like molecules which were evidently already loaded with amino acids during its structuring, this made possible a late origin of ARSs. All this corroborates the coevolution theory of the origin of the genetic code to the detriment of theories which would instead predict an early intervention of the action of ARSs in organizing the genetic code. Furthermore, the assembly of the GlyRS and LysRS protein domains in main phyletic lineages is itself at least evidence of the possibility that ancestral genes were assembled using pieces of genetic material that coded these protein domains. This is in accordance with the exon theory of genes which postulates that ancestral exons coded for protein domains or modules that were assembled to form the first genes. This theory is exemplified precisely in the evolution of both GlyRS and LysRS which occurred through the assembly of protein domains in the main phyletic lineages, as analyzed here. Furthermore, this late assembly of protein domains of these proteins into the two main phyletic lineages, i.e. a polyphyletic origin of both GlyRS and LysRS, appears to corroborate the progenote evolutionary stage for both LUCA and at least the first part of the evolutionary stages of the ancestor of bacteria and that of archaea. Indeed, this polyphyletic origin would imply that the genetic code was still evolving because at least two ARSs, i.e. proteins that make the genetic code possible today, were still evolving. This would imply that the evolutionary stages involved were characterized not by cells but by protocells, that is, by progenotes because this is precisely the definition of a progenote. This conclusion would be strengthened by the observation that both GlyRS and LysRS originating in the phyletic lineages leading to bacteria and archaea, would demonstrate that, more generally, proteins were most likely still in rapid and progressive evolution. Namely, a polyphyletic origin of proteins which would qualify at least the initial phase of the evolutionary stage of the ancestor of bacteria and that of archaea as stages belonging to the progenote.

A p- adic approach to the TSPO gene.

TSPO protein is known to be involved in various cellular functions and dysregulations of TSPO expression has been found to be associated with pathologies of different human diseases, including cardiovascular disease, cancer, neuroinflammatory, neurodegenerative, neoplastic disorders. However, there are limited studies in the literature on the effects of sequence variations in the TSPO gene on the function of the protein and their relationship with human diseases. Evaluating the pathogenicity of genetic variants is crucial in terms of prioritizing the functional importance and clinical use. Therefore, various in-silico prediction tools have been developed that combine different algorithms to predict the effects of sequence variations on protein functions or gene regulation. In this study, the p-adic distance approach in modeling the genetic code, proposed and developed by Dragovich and Dragovich, was discussed in order to obtain an alternative to the existing in-silico prediction tools. Dragovichs' approach is expressed as follows: A 5-adic space of codons is constructed and 5-adic and 2-adic distances between codons are taken into account. As a result, two codons with the smallest value of 5-adic and 2-adic distances are obtained, encoded for the same amino acid and stop signal. This model describes well the degeneration of the genetic code. This study combined the data obtained from in-silico prediction tools and used a bioinformatics approach to determine the functional relevance of coding SNPs in the TSPO. Overall, we evaluate the potential utility of Dragovichs' approach by comparing it with other existing prediction tools for variant classification and prioritization.

Noncoding translation mitigation.

Translation is pervasive outside of canonical coding regions, occurring in long noncoding RNAs, canonical untranslated regions and introns1-4, especially in ageing4-6, neurodegeneration5,7 and cancer8-10. Notably, the majority of tumour-specific antigens are results of noncoding translation11-13. Although the resulting polypeptides are often nonfunctional, translation of noncoding regions is nonetheless necessary for the birth of new coding sequences14,15. The mechanisms underlying the surveillance of translation in diverse noncoding regions and how escaped polypeptides evolve new functions remain unclear10,16-19. Functional polypeptides derived from annotated noncoding sequences often localize to membranes20,21. Here we integrate massively parallel analyses of more than 10,000 human genomic sequences and millions of random sequences with genome-wide CRISPR screens, accompanied by in-depth genetic and biochemical characterizations. Our results show that the intrinsic nucleotide bias in the noncoding genome and in the genetic code frequently results in polypeptides with a hydrophobic C-terminal tail, which is captured by the ribosome-associated BAG6 membrane protein triage complex for either proteasomal degradation or membrane targeting. By contrast, canonical proteins have evolved to deplete C-terminal hydrophobic residues. Our results reveal a fail-safe mechanism for the surveillance of unwanted translation from diverse noncoding regions and suggest a possible biochemical route for the preferential membrane localization of newly evolved proteins.

Hyperaccurate Ribosomes for Improved Genetic Code Reprogramming.

The reprogramming of the genetic code through the introduction of noncanonical amino acids (ncAAs) has enabled exciting advances in synthetic biology and peptide drug discovery. Ribosomes that function with high efficiency and fidelity are necessary for all of these efforts, but for challenging ncAAs, the competing processes of near-cognate readthrough and peptidyl-tRNA dropoff can be issues. Here we uncover the surprising extent of these competing pathways in the PURE translation system using mRNAs encoding peptides with affinity tags at the N- and C-termini. We also show that hyperaccurate or error restrictive ribosomes with mutations in ribosomal protein S12 lead to significant improvements in yield and fidelity in the context of both canonical AAs and a challenging α,α-disubstituted ncAA. Hyperaccurate ribosomes also improve yields for quadruplet codon readthrough for a tRNA containing an expanded anticodon stem-loop, although they are not able to eliminate triplet codon reading by this tRNA. The impressive improvements in fidelity and the simplicity of introducing this mutation alongside other efforts to engineer the translation apparatus make hyperaccurate ribosomes an important advance for synthetic biology.

Did Amino Acid Side Chain Reactivity Dictate the Composition and Timing of Aminoacyl-tRNA Synthetase Evolution?

The twenty amino acids in the standard genetic code were fixed prior to the last universal common ancestor (LUCA). Factors that guided this selection included establishment of pathways for their metabolic synthesis and the concomitant fixation of substrate specificities in the emerging aminoacyl-tRNA synthetases (aaRSs). In this conceptual paper, we propose that the chemical reactivity of some amino acid side chains (e.g., lysine, cysteine, homocysteine, ornithine, homoserine, and selenocysteine) delayed or prohibited the emergence of the corresponding aaRSs and helped define the amino acids in the standard genetic code. We also consider the possibility that amino acid chemistry delayed the emergence of the glutaminyl- and asparaginyl-tRNA synthetases, neither of which are ubiquitous in extant organisms. We argue that fundamental chemical principles played critical roles in fixation of some aspects of the genetic code pre- and post-LUCA.

Comprehensive study for BRCA1 and BRCA2 entire coding regions in breast cancer.

BACKGROUND: About 5-10% of incidences of breast cancers have been reported as a result of germline mutations of BRCA genes. However, the mutational spectrum of BRCA1 and BRCA2 genes among breast cancer Saudi women patients is inadequate at present. Therefore, the present study aimed to report the specific germinal mutation of BRCA1 and BRCA2 in the entire coding regions, to investigate the prevalence rate of BRCA1 & BRCA2 mutations among Saudi women and the effect of these mutations, both benign and malignant tumors. METHODOLOGY: A total of 270 tissue samples of benign and malignant breast tumors were collected from Saudi women patients, Riyadh, Saudi Arabia. Examination of BRCA1 and BRCA2 germline mutations was performed using heteroduplex DNA analysis (HDA) or single-stranded conformation analysis (SSCA). 177 breast cancer women with malignant tumors and 93 with benign tumors were enrolled in the study. A total of 62 out of 177 breast cancer patients carried a BRCA1 or BRCA2 mutation (54 BRCA1 and 8 BRCA2). The analysis was done using the Sanger sequence assay. RESULTS: Point and frameshift mutations through the entire coding area of the two genes indicated that all the mutations were germline alterations and of early-onset breast cancers. The mean ages of diagnosed breast cancer women for BRCA1 and BRCA2 mutation carriers were 36.3 (± 3.5) and 37.9 (± 3.7) years, whereas that of benign control was 35(± 2.5) years. CONCLUSION: Point and frameshift mutations across the entire coding region of BRCA1 and BRCA2 are responsible for many breast cancers cases.

A Genetically Encoded, Phage-Displayed Cyclic-Peptide Library.

Superior to linear peptides in biological activities, cyclic peptides are considered to have great potential as therapeutic agents. To identify cyclic-peptide ligands for therapeutic targets, phage-displayed peptide libraries in which cyclization is achieved by the covalent conjugation of cysteines have been widely used. To resolve drawbacks related to cysteine conjugation, we have invented a phage-display technique in which its displayed peptides are cyclized through a proximity-driven Michael addition reaction between a cysteine and an amber-codon-encoded Nϵ -acryloyl-lysine (AcrK). Using a randomized 6-mer library in which peptides were cyclized at two ends through a cysteine-AcrK linker, we demonstrated the successful selection of potent ligands for TEV protease and HDAC8. All selected cyclic peptide ligands showed 4- to 6-fold stronger affinity to their protein targets than their linear counterparts. We believe this approach will find broad applications in drug discovery.

Nonproteinogenic deep mutational scanning of linear and cyclic peptides.

High-resolution structure-activity analysis of polypeptides requires amino acid structures that are not present in the universal genetic code. Examination of peptide and protein interactions with this resolution has been limited by the need to individually synthesize and test peptides containing nonproteinogenic amino acids. We describe a method to scan entire peptide sequences with multiple nonproteinogenic amino acids and, in parallel, determine the thermodynamics of binding to a partner protein. By coupling genetic code reprogramming to deep mutational scanning, any number of amino acids can be exhaustively substituted into peptides, and single experiments can return all free energy changes of binding. We validate this approach by scanning two model protein-binding peptides with 21 diverse nonproteinogenic amino acids. Dense structure-activity maps were produced at the resolution of single aliphatic atom insertions and deletions. This permits rapid interrogation of interaction interfaces, as well as optimization of affinity, fine-tuning of physical properties, and systematic assessment of nonproteinogenic amino acids in binding and folding.

Structural basis for tRNA-dependent cysteine biosynthesis.

Cysteine can be synthesized by tRNA-dependent mechanism using a two-step indirect pathway, where O-phosphoseryl-tRNA synthetase (SepRS) catalyzes the ligation of a mismatching O-phosphoserine (Sep) to tRNACys followed by the conversion of tRNA-bounded Sep into cysteine by Sep-tRNA:Cys-tRNA synthase (SepCysS). In ancestral methanogens, a third protein SepCysE forms a bridge between the two enzymes to create a ternary complex named the transsulfursome. By combination of X-ray crystallography, SAXS and EM, together with biochemical evidences, here we show that the three domains of SepCysE each bind SepRS, SepCysS, and tRNACys, respectively, which mediates the dynamic architecture of the transsulfursome and thus enables a global long-range channeling of tRNACys between SepRS and SepCysS distant active sites. This channeling mechanism could facilitate the consecutive reactions of the two-step indirect pathway of Cys-tRNACys synthesis (tRNA-dependent cysteine biosynthesis) to prevent challenge of translational fidelity, and may reflect the mechanism that cysteine was originally added into genetic code.

Genetically Encoded Fluorescent RNA Sensor for Ratiometric Imaging of MicroRNA in Living Tumor Cells.

Light-up RNA aptamers are valuable tools for fluorescence imaging of RNA in living cells and thus for elucidating RNA functions and dynamics. However, no light-up RNA sensor has been reported for imaging of microRNAs (miRs) in mammalian cells. We report a novel genetically encoded RNA sensor for fluorescent imaging of miRs in living tumor cells using a light-up RNA aptamer that binds to sulforhodamine and separates it from a conjugated contact quencher. On the basis of the structural switching mechanism for molecular beacon, we show that the RNA sensor activates high-contrast fluorescence from the sulforhodamine-quencher conjugate when its stem-loop responsive motif hybridizes with target miR. The RNA sensor can be stably expressed within a designed tRNA scaffold in tumor cells and deliver light-up response to miR target. We also realize the RNA sensor for dual-emission, ratiometric imaging by coexpression of RNA sensor with GFP, enabling quantitative studies of target miR in living cells. Our design may provide a new paradigm for developing robust, sensitive light-up RNA sensors for RNA imaging applications.

Engineered Split-TET2 Enzyme for Inducible Epigenetic Remodeling.

The Ten-eleven translocation (TET) family of 5-methylcytosine (5mC) dioxygenases catalyze the conversion of 5mC into 5-hydroxymethylcytosine (5hmC) and further oxidized species to promote active DNA demethylation. Here we engineered a split-TET2 enzyme to enable temporal control of 5mC oxidation and subsequent remodeling of epigenetic states in mammalian cells. We further demonstrate the use of this chemically inducible system to dissect the correlation between DNA hydroxymethylation and chromatin accessibility in the mammalian genome. This chemical-inducible epigenome remodeling tool will find broad use in interrogating cellular systems without altering the genetic code, as well as in probing the epigenotype-phenotype relations in various biological systems.

Genetic code expansion in the mouse brain.

Site-specific incorporation of non-natural amino acids into proteins, via genetic code expansion with pyrrolysyl tRNA synthetase (PylRS) and tRNA(Pyl)CUA pairs (and their evolved derivatives) from Methanosarcina sp., forms the basis of powerful approaches to probe and control protein function in cells and invertebrate organisms. Here we demonstrate that adeno-associated viral delivery of these pairs enables efficient genetic code expansion in primary neuronal culture, organotypic brain slices and the brains of live mice.

Whisper mutations: cryptic messages within the genetic code.

Recent years have seen a great expansion in our understandings of how silent mutations can drive a disease and that mRNAs are not only mere messengers between the genome and the encoded proteins but also encompass regulatory activities. This review focuses on how silent mutations within open reading frames can affect the functional properties of the encoded protein. We describe how mRNAs exert control of cell biological processes governed by the encoded proteins via translation kinetics, protein folding, mRNA stability, spatio-temporal protein expression and by direct interactions with cellular factors. These examples illustrate how additional levels of information lie within the coding sequences and that the degenerative genetic code is not redundant and have co-evolved with the encoded proteins. Hence, so called synonymous mutations are not always silent but 'whisper'.

Genetic code expansion enables live-cell and super-resolution imaging of site-specifically labeled cellular proteins.

Methods to site-specifically and densely label proteins in cellular ultrastructures with small, bright, and photostable fluorophores would substantially advance super-resolution imaging. Recent advances in genetic code expansion and bioorthogonal chemistry have enabled the site-specific labeling of proteins. However, the efficient incorporation of unnatural amino acids into proteins and the specific, fluorescent labeling of the intracellular ultrastructures they form for subdiffraction imaging has not been accomplished. Two challenges have limited progress in this area: (i) the low efficiency of unnatural amino acid incorporation that limits labeling density and therefore spatial resolution and (ii) the uncharacterized specificity of intracellular labeling that will define signal-to-noise, and ultimately resolution, in imaging. Here we demonstrate the efficient production of cystoskeletal proteins (ß-actin and vimentin) containing bicyclo[6.1.0]nonyne-lysine at genetically defined sites. We demonstrate their selective fluorescent labeling with respect to the proteome of living cells using tetrazine-fluorophore conjugates, creating densely labeled cytoskeletal ultrastructures. STORM imaging of these densely labeled ultrastructures reveals subdiffraction features, including nuclear actin filaments. This work enables the site-specific, live-cell, fluorescent labeling of intracellular proteins at high density for super-resolution imaging of ultrastructural features within cells.

Genetic encoding of caged cysteine and caged homocysteine in bacterial and mammalian cells.

We report the genetic incorporation of caged cysteine and caged homocysteine into proteins in bacterial and mammalian cells. The genetic code of these cells was expanded with an engineered pyrrolysine tRNA/tRNA synthetase pair that accepts both light-activatable amino acids as substrates. Incorporation was validated by reporter assays, western blots, and mass spectrometry, and differences in incorporation efficiency were explained by molecular modeling of synthetase-amino acid interactions. As a proof-of-principle application, the genetic replacement of an active-site cysteine residue with a caged cysteine residue in Renilla luciferase led to a complete loss of enzyme activity; however, upon brief exposure to UV light, a >150-fold increase in enzymatic activity was observed, thus showcasing the applicability of the caged cysteine in live human cells. A simultaneously conducted genetic replacement with homocysteine yielded an enzyme with greatly reduced activity, thereby demonstrating the precise probing of a protein active site. These discoveries provide a new tool for the optochemical control of protein function in mammalian cells and expand the set of genetically encoded unnatural amino acids.

Multiple site-specific installations of Nε-monomethyl-L-lysine into histone proteins by cell-based and cell-free protein synthesis.

Lysine methylation is one of the important post-translational modifications of histones, and produces an N(ε) -mono-, di-, or trimethyllysine residues. Multiple and site-specific lysine methylations of histones are essential to define epigenetic statuses and control heterochromatin formation, DNA repair, and transcription regulation. A method was previously developed to build an analogue of N(ε)-monomethyllysine, with cysteine substituting for lysine. Here, we have developed a new method of preparing histones bearing multiple N(ε)-monomethyllysine residues at specified positions. Release factor 1-knockout (RFzero) Escherichia coli cells or a cell-free system based on the RFzero cell lysate was used for protein synthesis, as in RFzero cells UAG is redefined as a sense codon for non-canonical amino acids. During protein synthesis, a tert-butyloxycarbonyl-protected N(ε)-monomethyllysine analogue is ligated to Methanosarcina mazei pyrrolysine tRNA (tRNA(Pyl)) by M. mazei pyrrolysyl-tRNA synthetase mutants, and is translationally incorporated into one or more positions specified by the UAG codon. Protecting groups on the protein are then removed with trifluoroacetic acid to generate N(ε)-monomethyllysine residues. We installed N(ε)-monomethyllysine residues at positions 4, 9, 27, 36, and/or 79 of human histone H3. Each of the N(ε)-monomethyllysine residues within the produced histone H3 was recognized by its specific antibody. Furthermore, the antibody recognized the authentic N(ε)-monomethyllysine residue at position 27 better than the N(ε)-monomethyllysine analogue built with cysteine. Mass spectrometry analyses also confirmed the lysine modifications on the produced histone H3. Thus, our method enables the installation of authentic N(ε)-monomethyllysines at multiple positions within a protein for large-scale production.

Evolutionary analysis of DNA-protein-coding regions based on a genetic code cube metric.

The right estimation of the evolutionary distance between DNA or protein sequences is the cornerstone of the current phylogenetic analysis based on distance methods. Herein, it is demonstrated that the Manhattan distance (dw), weighted by the evolutionary importance of the nucleotide bases in the codon, is a naturally derived metric in the standard genetic code cube inserted into the three-dimensional Euclidean space. Based on the application of distance dw, a novel evolutionary model is proposed. This model includes insertion/deletion mutations that are very important for cancer studies, but usually discarded in classical evolutionary models. In this study, the new evolutionary model was applied to the phylogenetic analysis of the DNA protein-coding regions of 13 mammal mitochondrial genomes and of four cancer genetic- susceptibility genes (ATM, BRCA1, BRCA2 and p53) from nine mammals. The opossum (a marsupial) was used as an out-group species for both sets of sequences. The new evolutionary model yielded the correct topology, while the current models failed to separate the evolutionarily distant species of mouse and opossum.

iMir: an integrated pipeline for high-throughput analysis of small non-coding RNA data obtained by smallRNA-Seq.

BACKGROUND: Qualitative and quantitative analysis of small non-coding RNAs by next generation sequencing (smallRNA-Seq) represents a novel technology increasingly used to investigate with high sensitivity and specificity RNA population comprising microRNAs and other regulatory small transcripts. Analysis of smallRNA-Seq data to gather biologically relevant information, i.e. detection and differential expression analysis of known and novel non-coding RNAs, target prediction, etc., requires implementation of multiple statistical and bioinformatics tools from different sources, each focusing on a specific step of the analysis pipeline. As a consequence, the analytical workflow is slowed down by the need for continuous interventions by the operator, a critical factor when large numbers of datasets need to be analyzed at once. RESULTS: We designed a novel modular pipeline (iMir) for comprehensive analysis of smallRNA-Seq data, comprising specific tools for adapter trimming, quality filtering, differential expression analysis, biological target prediction and other useful options by integrating multiple open source modules and resources in an automated workflow. As statistics is crucial in deep-sequencing data analysis, we devised and integrated in iMir tools based on different statistical approaches to allow the operator to analyze data rigorously. The pipeline created here proved to be efficient and time-saving than currently available methods and, in addition, flexible enough to allow the user to select the preferred combination of analytical steps. We present here the results obtained by applying this pipeline to analyze simultaneously 6 smallRNA-Seq datasets from either exponentially growing or growth-arrested human breast cancer MCF-7 cells, that led to the rapid and accurate identification, quantitation and differential expression analysis of ~450 miRNAs, including several novel miRNAs and isomiRs, as well as identification of the putative mRNA targets of differentially expressed miRNAs. In addition, iMir allowed also the identification of ~70 piRNAs (piwi-interacting RNAs), some of which differentially expressed in proliferating vs growth arrested cells. CONCLUSION: The integrated data analysis pipeline described here is based on a reliable, flexible and fully automated workflow, useful to rapidly and efficiently analyze high-throughput smallRNA-Seq data, such as those produced by the most recent high-performance next generation sequencers. iMir is available at http://www.labmedmolge.unisa.it/inglese/research/imir.