Maxim V. Gerashchenko

Genome-wide ribosome profiling reveals complex translational regulation in response to oxidative stress

Information on unique and coordinated regulation of transcription and translation in response to stress is central to the understanding of cellular homeostasis. Here we used ribosome profiling coupled with next-generation sequencing to examine the interplay between transcription and translation under conditions of hydrogen peroxide treatment in Saccharomyces cerevisiae. Hydrogen peroxide treatment led to a massive and rapid increase in ribosome occupancy of short upstream ORFs, including those with non-AUG translational starts, and of the N-terminal regions of ORFs that preceded the transcriptional response. In addition, this treatment induced the synthesis of N-terminally extended proteins and elevated stop codon read-through and frameshift events. It also increased ribosome occupancy at the beginning of ORFs and potentially the duration of the elongation step. We identified proteins whose synthesis was regulated rapidly by hydrogen peroxide posttranscriptionally; however, for the majority of genes increased protein synthesis followed transcriptional regulation. These data define the landscape of genome-wide regulation of translation in response to hydrogen peroxide and suggest that potentiation (coregulation of the transcript level and translation) is a feature of oxidative stress.

Translation inhibitors cause abnormalities in ribosome profiling experiments

Abstract 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.

Genome analysis reveals insights into physiology and longevity of the Brandt’s bat Myotis brandtii

Bats account for one-fifth of mammalian species, are the only mammals with powered flight, and are among the few animals that echolocate. The insect-eating Brandt’s bat (Myotis brandtii) is the longest-lived bat species known to date (lifespan exceeds 40 years) and, at 4–8 g adult body weight, is the most extreme mammal with regard to disparity between body mass and longevity. Here we report sequencing and analysis of the Brandt’s bat genome and transcriptome, which suggest adaptations consistent with echolocation and hibernation, as well as altered metabolism, reproduction and visual function. Unique sequence changes in growth hormone and insulin-like growth factor 1 receptors are also observed. The data suggest that an altered growth hormone/insulin-like growth factor 1 axis, which may be common to other long-lived bat species, together with adaptations such as hibernation and low reproductive rate, contribute to the exceptional lifespan of the Brandt’s bat.

Methionine restriction extends lifespan of Drosophila melanogaster under conditions of low amino acid status

Reduced methionine (Met) intake can extend lifespan of rodents, but whether this regimen represents a general strategy for regulating aging has been controversial. Here we report that Met restriction extends lifespan in both fruit flies and yeast, and that this effect requires low amino acid status. Met restriction in Drosophila mimicks the effect of dietary restriction and is associated with decreased reproduction. However, under conditions of high amino acid status, Met restriction is ineffective and the trade-off between longevity and reproduction is not observed. Overexpression of InRDN or Tsc2 inhibits lifespan extension by Met restriction, suggesting the role of TOR signaling in the Met control of longevity. Overall, this study defines the specific roles of Met and amino acid imbalance in aging and suggests that Met restiction is a general strategy for lifespan extension.

Adaptations to a Subterranean Environment and Longevity Revealed by the Analysis of Mole Rat Genomes

Subterranean mammals spend their lives in dark, unventilated environments that are rich in carbon dioxide and ammonia and low in oxygen. Many of these animals are also long-lived and exhibit reduced aging-associated diseases, such as neurodegenerative disorders and cancer. We sequenced the genome of the Damaraland mole rat (DMR, Fukomys damarensis) and improved the genome assembly of the naked mole rat (NMR, Heterocephalus glaber). Comparative genome analyses, along with the transcriptomes of related subterranean rodents, revealed candidate molecular adaptations for subterranean life and longevity, including a divergent insulin peptide, expression of oxygen-carrying globins in the brain, prevention of high CO2-induced pain perception, and enhanced ammonia detoxification. Juxtaposition of the genomes of DMR and other more conventional animals with the genome of NMR revealed several truly exceptional NMR features: unusual thermogenesis, an aberrant melatonin system, pain insensitivity, and unique processing of 28S rRNA. Together, these genomes and transcriptomes extend our understanding of subterranean adaptations, stress resistance, and longevity.

CUG Start Codon Generates Thioredoxin/Glutathione Reductase Isoforms in Mouse Testes

Mammalian cytosolic and mitochondrial thioredoxin reductases are essential selenocysteine-containing enzymes that control thioredoxin functions. Thioredoxin/glutathione reductase (TGR) is a third member of this enzyme family. It has an additional glutaredoxin domain and shows highest expression in testes. Herein, we found that human and several other mammalian TGR genes lack any AUG codons that could function in translation initiation. Although mouse and rat TGRs have such codons, we detected protein sequences upstream of them by immunoblot assays and direct proteomic analyses. Further gene engineering and expression analyses demonstrated that a CUG codon, located upstream of the sequences previously thought to initiate translation, is the actual start codon in mouse TGR. The use of this codon relies on the Kozak consensus sequence and ribosome-scanning mechanism. However, CUG serves as an inefficient start codon that allows downstream initiation, thus generating two isoforms of the enzyme in vivo and in vitro. The use of CUG evolved in mammalian TGRs, and in some of these organisms, GUG is used instead. The newly discovered longer TGR form shows cytosolic localization in cultured cells and is expressed in spermatids in mouse testes. This study shows that CUG codon is used as an inefficient start codon to generate protein isoforms in mouse.

Lifespan extension conferred by endoplasmic reticulum secretory pathway deficiency requires induction of the unfolded protein response.

Cells respond to accumulation of misfolded proteins in the endoplasmic reticulum (ER) by activating the unfolded protein response (UPR) signaling pathway. The UPR restores ER homeostasis by degrading misfolded proteins, inhibiting translation, and increasing expression of chaperones that enhance ER protein folding capacity. Although ER stress and protein aggregation have been implicated in aging, the role of UPR signaling in regulating lifespan remains unknown. Here we show that deletion of several UPR target genes significantly increases replicative lifespan in yeast. This extended lifespan depends on a functional ER stress sensor protein, Ire1p, and is associated with constitutive activation of upstream UPR signaling. We applied ribosome profiling coupled with next generation sequencing to quantitatively examine translational changes associated with increased UPR activity and identified a set of stress response factors up-regulated in the long-lived mutants. Besides known UPR targets, we uncovered up-regulation of components of the cell wall and genes involved in cell wall biogenesis that confer resistance to multiple stresses. These findings demonstrate that the UPR is an important determinant of lifespan that governs ER stress and identify a signaling network that couples stress resistance to longevity.

Dynein is a motor for nuclear rotation while vimentin IFs is a “brake”

The positioning of the nucleus is achieved by two interconnected processes, anchoring and migration, both of which are controlled by cytoskeleton structures. Rotation is a special type of nuclear motility in many cell types, but its significance remains unclear. We used a vimentin‐null cell line, MFT‐16, which shows extensive nuclear rotation to study the phenomenon in detail. By selective disruption of cytoskeletal structures and video‐microscopic analysis, nuclear rotation was a microtubule‐dependent process that F‐actin partially impedes. The dynein–dynactin complex is responsible and inhibiting this motor by expression of a dominant negative mutant of its component P‐150 completely stops it. Nuclear rotation is powered by dynein associated with the nuclear envelope along stationary microtubules, centrosomes remaining immobile. We confirmed that vimentin IFs inhibit nuclear rotation, and variant proteins of the mutated wild type gene for vimentin that lacked considerable fragments of the N‐ and C‐terminal domains restored nuclear anchoring. Immunochemical analysis showed that these mutated IFs also bound plectin, arguing for a key role of this cytolinker protein in nuclear anchoring. It is proposed that this versatile machinery guarantees not only rotation and the correct location of a nucleus, but also its orientation in a cell.

Ribonuclease selection for ribosome profiling

Ribosome profiling has emerged as a powerful method to assess global gene translation, but methodological and analytical challenges often lead to inconsistencies across labs and model organisms. A critical issue in ribosome profiling is nuclease treatment of ribosome–mRNA complexes, as it is important to ensure both stability of ribosomal particles and complete conversion of polysomes to monosomes. We performed comparative ribosome profiling in yeast and mice with various ribonucleases including I, A, S7 and T1, characterized their cutting preferences, trinucleotide periodicity patterns and coverage similarities across coding sequences, and showed that they yield comparable estimations of gene expression when ribosome integrity is not compromised. However, ribosome coverage patterns of individual transcripts had little in common between the ribonucleases. We further examined their potency at converting polysomes to monosomes across other commonly used model organisms, including bacteria, nematodes and fruit flies. In some cases, ribonuclease treatment completely degraded ribosome populations. Ribonuclease T1 was the only enzyme that preserved ribosomal integrity while thoroughly converting polysomes to monosomes in all examined species. This study provides a guide for ribonuclease selection in ribosome profiling experiments across most common model systems.

Thiol Peroxidase Deficiency Leads to Increased Mutational Load and Decreased Fitness in Saccharomyces cerevisiae

Thiol peroxidases are critical enzymes in the redox control of cellular processes that function by reducing low levels of hydroperoxides and regulating redox signaling. These proteins were also shown to regulate genome stability, but how their dysfunction affects the actual mutations in the genome is not known. Saccharomyces cerevisiae has eight thiol peroxidases of glutathione peroxidase and peroxiredoxin families, and the mutant lacking all these genes (∆8) is viable. In this study, we employed two independent ∆8 isolates to analyze the genome-wide mutation spectrum that results from deficiency in these enzymes. Deletion of these genes was accompanied by a dramatic increase in point mutations, many of which clustered in close proximity and scattered throughout the genome, suggesting strong mutational bias. We further subjected multiple lines of wild-type and ∆8 cells to long-term mutation accumulation, followed by genome sequencing and phenotypic characterization. ∆8 lines showed a significant increase in nonrecurrent point mutations and indels. The original ∆8 cells exhibited reduced growth rate and decreased life span, which were further reduced in all ∆8 mutation accumulation lines. Although the mutation spectrum of the two independent isolates was different, similar patterns of gene expression were observed, suggesting the direct contribution of thiol peroxidases to the observed phenotypes. Expression of a single thiol peroxidase could partially restore the growth phenotype of ∆8 cells. This study shows how deficiency in nonessential, yet critical and conserved oxidoreductase function, leads to increased mutational load and decreased fitness.