Translation inhibitors cause abnormalities in ribosome profiling experiments

Ribosome profiling and high-throughput sequencing provide unprecedented opportunities for the analysis of mRNA translation. Using this novel method, several studies have demonstrated the widespread role of short upstream reading frames in translational control as well as slower elongation at the beginning of open reading frames in response to stress. Based on the initial studies, the importance of adding or omitting translation inhibitors, such as cycloheximide, was noted as it markedly affected ribosome coverage profiles. For that reason, many recent studies omitted translation inhibitors in the culture medium. Here, we investigate the influence of ranging cycloheximide concentrations on ribosome profiles in Saccharomyces cerevisiae and demonstrate that increasing the drug concentration can overcome some of the artifacts. We subjected cells to various manipulations and show that neither oxidative stress nor heat shock nor amino acid starvation affect translation elongation. Instead, the observations in the initial studies are the result of cycloheximide-inflicted artifacts. Likewise, we find little support for short upstream reading frames to be involved in widespread protein synthesis regulation under stress conditions. Our study highlights the need for better standardization of ribosome profiling methods

Aging: progressive decline in fitness due to the rising deleteriome adjusted by genetic, environmental, and stochastic processes

Summary Different theories posit that aging is caused by molecular damage, genetic programs, continued development, hyperfunction, antagonistic pleiotropy alleles, mutations, trade‐offs, incomplete repair, etc. Here, I discuss that these ideas can be conceptually unified as they capture particular facets of aging, while being incomplete. Their respective deleterious effects impact fitness at different levels of biological organization, adjusting progression through aging, rather than causing it. Living is associated with a myriad of deleterious processes, both random and deterministic, which are caused by imperfectness, exhibit cumulative properties, and represent the indirect effects of biological functions at all levels, from simple molecules to systems. From this, I derive the deleteriome, which encompasses cumulative deleterious age‐related changes and represents the biological age. The organismal deleteriome consists of the deleteriomes of cells, organs, and systems, which change along roughly synchronized trajectories and may be assessed through biomarkers of aging. Aging is then a progressive decline in fitness due to the increasing deleteriome, adjusted by genetic, environmental, and stochastic processes. This model allows integration of diverse aging concepts, provides insights into the nature of aging, and suggests how lifespan may be adjusted during evolution and in experimental models

Alternative first exon splicing regulates subcellular distribution of methionine sulfoxide reductases

BACKGROUND: Methionine sulfoxide reduction is an important protein repair pathway that protects against oxidative stress, controls protein function and has a role in regulation of aging. There are two enzymes that reduce stereospecifically oxidized methionine residues: MsrA (methionine-S-sulfoxide reductase) and MsrB (methionine-R-sulfoxide reductase). In many organisms, these enzymes are targeted to various cellular compartments. In mammals, a single MsrA gene is known, however, its product is present in cytosol, nucleus, and mitochondria. In contrast, three mammalian MsrB genes have been identified whose products are located in different cellular compartments. RESULTS: In the present study, we identified and characterized alternatively spliced forms of mammalian MsrA. In addition to the previously known variant containing an N-terminal mitochondrial signal peptide and distributed between mitochondria and cytosol, a second mouse and human form was detected in silico. This form, MsrA(S), was generated using an alternative first exon. MsrA(S) was enzymatically active and was present in cytosol and nucleus in transfected cells, but occurred below detection limits in tested mouse tissues. The third alternative form lacked the active site and could not be functional. In addition, we found that mitochondrial and cytosolic forms of both MsrA and MsrB in Drosophila could be generated by alternative first exon splicing. CONCLUSION: Our data suggest conservation of alternative splicing to regulate subcellular distribution of methionine sulfoxide reductases

The microbial selenoproteome of the Sargasso Sea

BACKGROUND: Selenocysteine (Sec) is a rare amino acid which occurs in proteins in major domains of life. It is encoded by TGA, which also serves as the signal for termination of translation, precluding identification of selenoprotein genes by available annotation tools. Information on full sets of selenoproteins (selenoproteomes) is essential for understanding the biology of selenium. Herein, we characterized the selenoproteome of the largest microbial sequence dataset, the Sargasso Sea environmental genome project. RESULTS: We identified 310 selenoprotein genes that clustered into 25 families, including 101 new selenoprotein genes that belonged to 15 families. Most of these proteins were predicted redox proteins containing catalytic selenocysteines. Several bacterial selenoproteins previously thought to be restricted to eukaryotes were detected by analyzing eukaryotic and bacterial SECIS elements, suggesting that eukaryotic and bacterial selenoprotein sets partially overlapped. The Sargasso Sea microbial selenoproteome was rich in selenoproteins and its composition was different from that observed in the combined set of completely sequenced genomes, suggesting that these genomes do not accurately represent the microbial selenoproteome. Most detected selenoproteins occurred sporadically compared to the widespread presence of their cysteine homologs, suggesting that many selenoproteins recently evolved from cysteine-containing homologs. CONCLUSIONS: This study yielded the largest selenoprotein dataset to date, doubled the number of prokaryotic selenoprotein families and provided insights into forces that drive selenocysteine evolution

Reduced reliance on the trace element selenium during evolution of mammals

Evolution from fish to mammals was accompanied by decreased use of selenocysteine, raising questions about the need for selenium dietary supplements when pathology is not imminent

SECISearch3 and Seblastian: new tools for prediction of SECIS elements and selenoproteins

Selenoproteins are proteins containing an uncommon amino acid selenocysteine (Sec). Sec is inserted by a specific translational machinery that recognizes a stem-loop structure, the SECIS element, at the 3′ UTR of selenoprotein genes and recodes a UGA codon within the coding sequence. As UGA is normally a translational stop signal, selenoproteins are generally misannotated and designated tools have to be developed for this class of proteins. Here, we present two new computational methods for selenoprotein identification and analysis, which we provide publicly through the web servers at http://gladyshevlab.org/SelenoproteinPredictionServer or http://seblastian.crg.es. SECISearch3 replaces its predecessor SECISearch as a tool for prediction of eukaryotic SECIS elements. Seblastian is a new method for selenoprotein gene detection that uses SECISearch3 and then predicts selenoprotein sequences encoded upstream of SECIS elements. Seblastian is able to both identify known selenoproteins and predict new selenoproteins. By applying these tools to diverse eukaryotic genomes, we provide a ranked list of newly predicted selenoproteins together with their annotated cysteine-containing homologues. An analysis of a representative candidate belonging to the AhpC family shows how the use of Sec in this protein evolved in bacterial and eukaryotic lineages

SelenoDB 1.0 : A Database of Selenoprotein Genes, Proteins and SECIS Elements

Selenoproteins are a diverse group of proteins usually misidentified and misannotated in sequence databases. The presence of an in-frame UGA (stop) codon in the coding sequence of selenoprotein genes precludes their identification and correct annotation. The in-frame UGA codons are recoded to cotranslationally incorporate selenocysteine, a rare selenium-containing amino acid. The development of ad hoc experimental and, more recently, computational approaches have allowed the efficient identification and characterization of the selenoproteomes of a growing number of species. Today, dozens of selenoprotein families have been described and more are being discovered in recently sequenced species, but the correct genomic annotation is not available for the majority of these genes. SelenoDB is a long-term project that aims to provide, through the collaborative effort of experimental and computational researchers, automatic and manually curated annotations of selenoprotein genes, proteins and SECIS elements. Version 1.0 of the database includes an initial set of eukaryotic genomic annotations, with special emphasis on the human selenoproteome, for immediate inspection by selenium researchers or incorporation into more general databases. SelenoDB is freely available at http://www.selenodb.org