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The p53 pathway is pivotal in tumor suppression. Cellular p53 activity is subject to tight regulation, in which the two related proteins Mdm2 and Mdm4 have major roles. The delicate interplay between the levels of Mdm2, Mdm4 and p53 is crucial for maintaining proper cellular homeostasis. microRNAs (miRNAs) are short non-coding RNAs that downregulate the level and translatability of specific target mRNAs. We report that miR-661, a primate-specific miRNA, can target both Mdm2 and Mdm4 mRNA in a cell type-dependent manner. miR-661 interacts with Mdm2 and Mdm4 RNA within living cells. The inhibitory effect of miR-661 is more prevalent on Mdm2 than on Mdm4. Interestingly, the predicted miR-661 targets in both mRNAs reside mainly within Alu elements, suggesting a primate-specific mechanism for regulatory diversification during evolution. Downregulation of Mdm2 and Mdm4 by miR-661 augments p53 activity and inhibits cell cycle progression in p53-proficient cells. Correspondingly, low miR-661 expression correlates with bad outcome in breast cancers that typically express wild-type p53. In contrast, the miR-661 locus tends to be amplified in tumors harboring p53 mutations, and miR-661 promotes migration of cells derived from such tumors. Thus, miR-661 may either suppress or promote cancer aggressiveness, depending on p53 status. [ABSTRACT FROM AUTHOR]

Aberrant oncogene activation induces cellular senescence, an irreversible growth arrest that acts as a barrier against tumorigenesis. To identify microRNAs (miRNAs) involved in oncogene-induced senescence, we examined the expression of miRNAs in primary human TIG3 fibroblasts after constitutive activation of B-RAF. Among the regulated miRNAs, both miR-34a and miR-146a were strongly induced during senescence. Although members of the miR-34 family are known to be transcriptionally regulated by p53, we find that miR-34a is regulated independently of p53 during oncogene-induced senescence. Instead, upregulation of miR-34a is mediated by the ETS family transcription factor, ELK1. During senescence, miR-34a targets the important proto-oncogene MYC and our data suggest that miR-34a thereby coordinately controls a set of cell cycle regulators. Hence, in addition to its integration in the p53 pathway, we show that alternative cancer-related pathways regulate miR-34a, emphasising its significance as a tumour suppressor. [ABSTRACT FROM AUTHOR]

The genetic diversity of the gut microbiota has a central role in host health. Here, we created pangenomes for 728 human gut prokaryotic species, quadrupling the genes of strain-specific genomes. Each of these species has a core set of a thousand genes, differing even between closely related species, and an accessory set of genes unique to the different strains. Functional analysis shows high strain variability associates with sporulation, whereas low variability is linked with antibiotic resistance. We further map the antibiotic resistome across the human gut population and find 237 cases of extreme resistance even to last-resort antibiotics, with a predominance among Enterobacteriaceae. Lastly, the presence of specific genes in the microbiota relates to host age and sex. Our study underscores the genetic complexity of the human gut microbiota, emphasizing its significant implications for host health. The pangenomes and antibiotic resistance map constitute a valuable resource for further research., Competing Interests: Declaration of interests E.S. is a paid consultant to Pheno.AI, Ltd., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Leishmania is the causative agent of cutaneous and visceral diseases affecting millions of individuals worldwide. Pseudouridine (Ψ), the most abundant modification on rRNA, changes during the parasite life cycle. Alterations in the level of a specific Ψ in helix 69 (H69) affected ribosome function. To decipher the molecular mechanism of this phenotype, we determine the structure of ribosomes lacking the single Ψ and its parental strain at ∼2.4-3 Å resolution using cryo-EM. Our findings demonstrate the significance of a single Ψ on H69 to its structure and the importance for its interactions with helix 44 and specific tRNAs. Our study suggests that rRNA modification affects translation of mRNAs carrying codon bias due to selective accommodation of tRNAs by the ribosome. Based on the high-resolution structures, we propose a mechanism explaining how the ribosome selects specific tRNAs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The mycobiota are a critical part of the gut microbiome, but host-fungal interactions and specific functional contributions of commensal fungi to host fitness remain incompletely understood. Here, we report the identification of a new fungal commensal, Kazachstania heterogenica var. weizmannii, isolated from murine intestines. K. weizmannii exposure prevented Candida albicans colonization and significantly reduced the commensal C. albicans burden in colonized animals. Following immunosuppression of C. albicans colonized mice, competitive fungal commensalism thereby mitigated fatal candidiasis. Metagenome analysis revealed K. heterogenica or K. weizmannii presence among human commensals. Our results reveal competitive fungal commensalism within the intestinal microbiota, independent of bacteria and immune responses, that could bear potential therapeutic value for the management of C. albicans-mediated diseases., (© 2024 Sekeresova Kralova et al.)

Some drugs increase the mutation rate of their target pathogen, a potentially concerning mechanism as the pathogen might evolve faster toward an undesired phenotype. We suggest a four-step assessment of evolutionary safety for the approval of such treatments., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Lobinska et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Aneuploidy is common in eukaryotes, often leading to decreased fitness. However, evidence from fungi and human tumur cells suggests that specific aneuploidies can be beneficial under stressful conditions and facilitate adaptation. In a previous evolutionary experiment with yeast, populations evolving under heat stress became aneuploid, only to later revert to euploidy after beneficial mutations accumulated. It was therefore suggested that aneuploidy is a "stepping stone" on the path to adaptation. Here, we test this hypothesis. We use Bayesian inference to fit an evolutionary model with both aneuploidy and mutation to the experimental results. We then predict the genotype frequency dynamics during the experiment, demonstrating that most of the evolved euploid population likely did not descend from aneuploid cells, but rather from the euploid wild-type population. Our model shows how the beneficial mutation supply-the product of population size and beneficial mutation rate-determines the evolutionary dynamics: with low supply, much of the evolved population descends from aneuploid cells; but with high supply, beneficial mutations are generated fast enough to outcompete aneuploidy due to its inherent fitness cost. Our results suggest that despite its potential fitness benefits under stress, aneuploidy can be an evolutionary "diversion" rather than a "stepping stone": it can delay, rather than facilitate, the adaptation of the population, and cells that become aneuploid may leave less descendants compared to cells that remain diploid., (© The Author(s) 2024. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

Diabetes and associated comorbidities are a global health threat on the rise. We conducted a six-month dietary intervention in pre-diabetic individuals (NCT03222791), to mitigate the hyperglycemia and enhance metabolic health. The current work explores early diabetes markers in the 200 individuals who completed the trial. We find 166 of 2,803 measured features, including oral and gut microbial species and pathways, serum metabolites and cytokines, show significant change in response to a personalized postprandial glucose-targeting diet or the standard of care Mediterranean diet. These changes include established markers of hyperglycemia as well as novel features that can now be investigated as potential therapeutic targets. Our results indicate the microbiome mediates the effect of diet on glycemic, metabolic and immune measurements, with gut microbiome compositional change explaining 12.25% of serum metabolites variance. Although the gut microbiome displays greater compositional changes compared to the oral microbiome, the oral microbiome demonstrates more changes at the genetic level, with trends dependent on environmental richness and species prevalence in the population. In conclusion, our study shows dietary interventions can affect the microbiome, cardiometabolic profile and immune response of the host, and that these factors are well associated with each other, and can be harnessed for new therapeutic modalities., (© 2023. Springer Nature Limited.)

The mutation rate plays an important role in adaptive evolution. It can be modified by mutator and anti-mutator alleles. Recent empirical evidence hints that the mutation rate may vary among genetically identical individuals: evidence from bacteria suggests that the mutation rate can be affected by expression noise of a DNA repair protein and potentially also by translation errors in various proteins. Importantly, this non-genetic variation may be heritable via a transgenerational epigenetic mode of inheritance, giving rise to a mutator phenotype that is independent from mutator alleles. Here, we investigate mathematically how the rate of adaptive evolution is affected by the rate of mutation rate phenotype switching. We model an asexual population with two mutation rate phenotypes, non-mutator and mutator. An offspring may switch from its parental phenotype to the other phenotype. We find that switching rates that correspond to so-far empirically described non-genetic systems of inheritance of the mutation rate lead to higher rates of adaptation on both artificial and natural fitness landscapes. These switching rates can maintain within the same individuals both a mutator phenotype and intermediary mutations, a combination that facilitates adaptation. Moreover, non-genetic inheritance increases the proportion of mutators in the population, which in turn increases the probability of hitchhiking of the mutator phenotype with adaptive mutations. This in turns facilitates the acquisition of additional adaptive mutations. Our results rationalize recently observed noise in the expression of proteins that affect the mutation rate and suggest that non-genetic inheritance of this phenotype may facilitate evolutionary adaptive processes., Competing Interests: Conflicts of interest: The author(s) declare no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of The Genetics Society of America.)

Nucleoside analogs are a major class of antiviral drugs. Some act by increasing the viral mutation rate causing lethal mutagenesis of the virus. Their mutagenic capacity, however, may lead to an evolutionary safety concern. We define evolutionary safety as a probabilistic assurance that the treatment will not generate an increased number of mutants. We develop a mathematical framework to estimate the total mutant load produced with and without mutagenic treatment. We predict rates of appearance of such virus mutants as a function of the timing of treatment and the immune competence of patients, employing realistic assumptions about the vulnerability of the viral genome and its potential to generate viable mutants. We focus on the case study of Molnupiravir, which is an FDA-approved treatment against Coronavirus Disease-2019 (COVID-19). We estimate that Molnupiravir is narrowly evolutionarily safe, subject to the current estimate of parameters. Evolutionary safety can be improved by restricting treatment with this drug to individuals with a low immunological clearance rate and, in future, by designing treatments that lead to a greater increase in mutation rate. We report a simple mathematical rule to determine the fold increase in mutation rate required to obtain evolutionary safety that is also applicable to other pathogen-treatment combinations., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Lobinska et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Cancer-microbe associations have been explored for centuries, but cancer-associated fungi have rarely been examined. Here, we comprehensively characterize the cancer mycobiome within 17,401 patient tissue, blood, and plasma samples across 35 cancer types in four independent cohorts. We report fungal DNA and cells at low abundances across many major human cancers, with differences in community compositions that differ among cancer types, even when accounting for technical background. Fungal histological staining of tissue microarrays supported intratumoral presence and frequent spatial association with cancer cells and macrophages. Comparing intratumoral fungal communities with matched bacteriomes and immunomes revealed co-occurring bi-domain ecologies, often with permissive, rather than competitive, microenvironments and distinct immune responses. Clinically focused assessments suggested prognostic and diagnostic capacities of the tissue and plasma mycobiomes, even in stage I cancers, and synergistic predictive performance with bacteriomes., Competing Interests: Declaration of interests G.D.S.-P. and R.K. are inventors on a US patent application (PCT/US2019/059647) submitted by The Regents of the University of California and licensed by Micronoma; that application covers methods of diagnosing and treating cancer using multi-domain microbial biomarkers in blood and cancer tissues. G.D.S.-P. and R.K. are founders of and report stock interest in Micronoma. G.D.S.-P. has filed several additional US patent applications on cancer bacteriome and mycobiome diagnostics that are owned by The Regents of the University of California. R.K. additionally is a member of the scientific advisory board for GenCirq, holds an equity interest in GenCirq, and can receive reimbursements for expenses up to US $5,000 per year. S.W. is an employee of Micronoma. R.S. received a grant from Merck EMD Serono, is a member of the scientific advisory board for Micronoma and reports stock interest in Micronoma, CuResponse, and Biomica, and is a paid adviser to Biomica, CuResponse, and BiomX. R.S., Y.P., I.L., and L.N.-H. are co-inventors on an Israeli provisional patent application (#284860) submitted by Yeda Research and Development, the Weizmann Institute of Science, that covers methods of diagnosing and treating cancer using mycobial biomarkers in cancer tissues., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Fitness landscape mapping and the prediction of evolutionary trajectories on these landscapes are major tasks in evolutionary biology research. Evolutionary dynamics is tightly linked to the landscape topography, but this relation is not straightforward. Here, we analyze a fitness landscape of a yeast tRNA gene, previously measured under four different conditions. We find that the wild type allele is sub-optimal, and 8-10% of its variants are fitter. We rule out the possibilities that the wild type is fittest on average on these four conditions or located on a local fitness maximum. Notwithstanding, we cannot exclude the possibility that the wild type might be fittest in some of the many conditions in the complex ecology that yeast lives at. Instead, we find that the wild type is mutationally robust ("flat"), while more fit variants are typically mutationally fragile. Similar observations of mutational robustness or flatness have been so far made in very few cases, predominantly in viral genomes., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

The COVID-19 pandemic demonstrated that the process of global vaccination against a novel virus can be a prolonged one. Social distancing measures, that are initially adopted to control the pandemic, are gradually relaxed as vaccination progresses and population immunity increases. The result is a prolonged period of high disease prevalence combined with a fitness advantage for vaccine-resistant variants, which together lead to a considerably increased probability for vaccine escape. A spatial vaccination strategy is proposed that has the potential to dramatically reduce this risk. Rather than dispersing the vaccination effort evenly throughout a country, distinct geographic regions of the country are sequentially vaccinated, quickly bringing each to effective herd immunity. Regions with high vaccination rates will then have low infection rates and vice versa. Since people primarily interact within their own region, spatial vaccination reduces the number of encounters between infected individuals (the source of mutations) and vaccinated individuals (who facilitate the spread of vaccine-resistant strains). Thus, spatial vaccination may help mitigate the global risk of vaccine-resistant variants., Competing Interests: The authors have declared that no competing interests exist.

Trypanosoma brucei, the parasite that causes sleeping sickness, cycles between an insect and a mammalian host. However, the effect of RNA modifications such as pseudouridinylation on its ability to survive in these two different host environments is unclear. Here, two genome-wide approaches were applied for mapping pseudouridinylation sites (Ψs) on small nucleolar RNA (snoRNA), 7SL RNA, vault RNA, and tRNAs from T. brucei. We show using HydraPsiSeq and RiboMeth-seq that the Ψ on C/D snoRNA guiding 2'-O-methylation increased the efficiency of the guided modification on its target, rRNA. We found differential levels of Ψs on these noncoding RNAs in the two life stages (insect host and mammalian host) of the parasite. Furthermore, tRNA isoform abundance and Ψ modifications were characterized in these two life stages demonstrating stage-specific regulation. We conclude that the differential Ψ modifications identified here may contribute to modulating the function of noncoding RNAs involved in rRNA processing, rRNA modification, protein synthesis, and protein translocation during cycling of the parasite between its two hosts., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The greatest hope for a return to normalcy following the COVID-19 pandemic is worldwide vaccination. Yet, a relaxation of social distancing that allows increased transmissibility, coupled with selection pressure due to vaccination, will probably lead to the emergence of vaccine resistance. We analyse the evolutionary dynamics of COVID-19 in the presence of dynamic contact reduction and in response to vaccination. We use infection and vaccination data from six different countries. We show that under slow vaccination, resistance is very likely to appear even if social distancing is maintained. Under fast vaccination, the emergence of mutants can be prevented if social distancing is maintained during vaccination. We analyse multiple human factors that affect the evolutionary potential of the virus, including the extent of dynamic social distancing, vaccination campaigns, vaccine design, boosters and vaccine hesitancy. We provide guidelines for policies that aim to minimize the probability of emergence of vaccine-resistant variants., (© 2022. The Author(s), under exclusive licence to Springer Nature Limited.)

The tRNA pool determines the efficiency, throughput, and accuracy of translation. Previous studies have identified dynamic changes in the tRNA (transfer RNA) supply and mRNA (messenger RNA) demand during cancerous proliferation. Yet dynamic changes may also occur during physiologically normal proliferation, and these are less well characterized. We examined the tRNA and mRNA pools of T cells during their vigorous proliferation and differentiation upon triggering their antigen receptor. We observed a global signature of switch in demand for codons at the early proliferation phase of the response, accompanied by corresponding changes in tRNA expression levels. In the later phase, upon differentiation, the response of the tRNA pool relaxed back to the basal level, potentially restraining excessive proliferation. Sequencing of tRNAs allowed us to evaluate their diverse base-modifications. We found that two types of tRNA modifications, wybutosine and ms 2 t6A, are reduced dramatically during T cell activation. These modifications occur in the anticodon loops of two tRNAs that decode "slippery codons," which are prone to ribosomal frameshifting. Attenuation of these frameshift-protective modifications is expected to increase the potential for proteome-wide frameshifting during T cell proliferation. Indeed, human cell lines deleted of a wybutosine writer showed increased ribosomal frameshifting, as detected with an HIV gag-pol frameshifting site reporter. These results may explain HIV's specific tropism toward proliferating T cells since it requires ribosomal frameshift exactly on the corresponding codon for infection. The changes in tRNA expression and modifications uncover a layer of translation regulation during T cell proliferation and expose a potential tradeoff between cellular growth and translation fidelity., Competing Interests: The authors declare no competing interest.

Stochastic transition of cancer cells between drug-sensitive and drug-tolerant persister phenotypes has been proposed to play a key role in non-genetic resistance to therapy. Yet, we show here that cancer cells actually possess a highly stable inherited chance to persist (CTP) during therapy. This CTP is non-stochastic, determined pre-treatment and has a unimodal distribution ranging from 0 to almost 100%. Notably, CTP is drug specific. We found that differential serine/threonine phosphorylation of the insulin receptor substrate 1 (IRS1) protein determines the CTP of lung and of head and neck cancer cells under epidermal growth factor receptor inhibition, both in vitro and in vivo. Indeed, the first-in-class IRS1 inhibitor NT219 was highly synergistic with anti-epidermal growth factor receptor therapy across multiple in vitro and in vivo models. Elucidation of drug-specific mechanisms that determine the degree and stability of cellular CTP may establish a framework for the elimination of cancer persisters, using new rationally designed drug combinations., (© 2021. The Author(s), under exclusive licence to Springer Nature America, Inc.)

RNA splicing is a key process in eukaryotic gene expression, in which an intron is spliced out of a pre-mRNA molecule to eventually produce a mature mRNA. Most intron-containing genes are constitutively spliced, hence efficient splicing of an intron is crucial for efficient regulation of gene expression. Here we use a large synthetic oligo library of ~20,000 variants to explore how different intronic sequence features affect splicing efficiency and mRNA expression levels in S. cerevisiae. Introns are defined by three functional sites, the 5' donor site, the branch site, and the 3' acceptor site. Using a combinatorial design of synthetic introns, we demonstrate how non-consensus splice site sequences in each of these sites affect splicing efficiency. We then show that S. cerevisiae splicing machinery tends to select alternative 3' splice sites downstream of the original site, and we suggest that this tendency created a selective pressure, leading to the avoidance of cryptic splice site motifs near introns' 3' ends. We further use natural intronic sequences from other yeast species, whose splicing machineries have diverged to various extents, to show how intron architectures in the various species have been adapted to the organism's splicing machinery. We suggest that the observed tendency for cryptic splicing is a result of a loss of a specific splicing factor, U2AF1. Lastly, we show that synthetic sequences containing two introns give rise to alternative RNA isoforms in S. cerevisiae, demonstrating that merely a synthetic fusion of two introns might be suffice to facilitate alternative splicing in yeast. Our study reveals novel mechanisms by which introns are shaped in evolution to allow cells to regulate their transcriptome. In addition, it provides a valuable resource to study the regulation of constitutive and alternative splicing in a model organism., Competing Interests: The authors have declared that no competing interests exist.

Different subsets of the tRNA pool in human cells are expressed in different cellular conditions. The 'proliferation-tRNAs' are induced upon normal and cancerous cell division, while the 'differentiation-tRNAs' are active in non-dividing, differentiated cells. Here we examine the essentiality of the various tRNAs upon cellular growth and arrest. We established a CRISPR-based editing procedure with sgRNAs that each target a tRNA family. We measured tRNA essentiality for cellular growth and found that most proliferation-tRNAs are essential compared to differentiation- tRNAs in rapidly growing cell lines. Yet in more slowly dividing lines, the differentiation-tRNAs were more essential. In addition, we measured the essentiality of each tRNA family upon response to cell cycle arresting signals. Here we detected a more complex behavior with both proliferation-tRNAs and differentiation tRNAs showing various levels of essentiality. These results provide the so-far most comprehensive functional characterization of human tRNAs with intricate roles in various cellular states., Competing Interests: NA, IF, YM, OD, YP, RR No competing interests declared, (© 2020, Aharon-Hefetz et al.)

Tracing evolutionary processes that lead to fixation of genomic variation in wild bacterial populations is a prime challenge in molecular evolution. In particular, the relative contribution of horizontal gene transfer (HGT) vs. de novo mutations during adaptation to a new environment is poorly understood. To gain a better understanding of the dynamics of HGT and its effect on adaptation, we subjected several populations of competent Bacillus subtilis to a serial dilution evolution on a high-salt-containing medium, either with or without foreign DNA from diverse pre-adapted or naturally salt tolerant species. Following 504 generations of evolution, all populations improved growth yield on the medium. Sequencing of evolved populations revealed extensive acquisition of foreign DNA from close Bacillus donors but not from more remote donors. HGT occurred in bursts, whereby a single bacterial cell appears to have acquired dozens of fragments at once. In the largest burst, close to 2% of the genome has been replaced by HGT. Acquired segments tend to be clustered in integration hotspots. Other than HGT, genomes also acquired spontaneous mutations. Many of these mutations occurred within, and seem to alter, the sequence of flagellar proteins. Finally, we show that, while some HGT fragments could be neutral, others are adaptive and accelerate evolution., (Copyright © 2020 by the Genetics Society of America.)

Programmed ribosomal frameshifting (PRF) is the controlled slippage of the translating ribosome to an alternative frame. This process is widely employed by human viruses such as HIV and SARS coronavirus and is critical for their replication. Here, we developed a high-throughput approach to assess the frameshifting potential of a sequence. We designed and tested >12,000 sequences based on 15 viral and human PRF events, allowing us to systematically dissect the rules governing ribosomal frameshifting and discover novel regulatory inputs based on amino acid properties and tRNA availability. We assessed the natural variation in HIV gag-pol frameshifting rates by testing >500 clinical isolates and identified subtype-specific differences and associations between viral load in patients and the optimality of PRF rates. We devised computational models that accurately predict frameshifting potential and frameshifting rates, including subtle differences between HIV isolates. This approach can contribute to the development of antiviral agents targeting PRF.

Most human genes are alternatively spliced, allowing for a large expansion of the proteome. The multitude of regulatory inputs to splicing limits the potential to infer general principles from investigating native sequences. Here, we create a rationally designed library of >32,000 splicing events to dissect the complexity of splicing regulation through systematic sequence alterations. Measuring RNA and protein splice isoforms allows us to investigate both cause and effect of splicing decisions, quantify diverse regulatory inputs and accurately predict (R 2  = 0.73-0.85) isoform ratios from sequence and secondary structure. By profiling individual cells, we measure the cell-to-cell variability of splicing decisions and show that it can be encoded in the DNA and influenced by regulatory inputs, opening the door for a novel, single-cell perspective on splicing regulation.

Splicing expands, reshapes, and regulates the transcriptome of eukaryotic organisms. Despite its importance, key questions remain unanswered, including the following: Can splicing evolve when organisms adapt to new challenges? How does evolution optimize inefficiency of introns' splicing and of the splicing machinery? To explore these questions, we evolved yeast cells that were engineered to contain an inefficiently spliced intron inside a gene whose protein product was under selection for an increased expression level. We identified a combination of mutations in Cis (within the gene of interest) and in Trans (in mRNA-maturation machinery). Surprisingly, the mutations in Cis resided outside of known intronic functional sites and improved the intron's splicing efficiency potentially by easing tight mRNA structures. One of these mutations hampered a protein's domain that was not under selection, demonstrating the evolutionary flexibility of multi-domain proteins as one domain functionality was improved at the expense of the other domain. The Trans adaptations resided in two proteins, Npl3 and Gbp2, that bind pre-mRNAs and are central to their maturation. Interestingly, these mutations either increased or decreased the affinity of these proteins to mRNA, presumably allowing faster spliceosome recruitment or increased time before degradation of the pre-mRNAs, respectively. Altogether, our work reveals various mechanistic pathways toward optimizations of intron splicing to ultimately adapt gene expression patterns to novel demands., Competing Interests: The authors have declared that no competing interests exist.

The translation machinery and the genes it decodes co-evolved to achieve production throughput and accuracy. Nonetheless, translation errors are frequent, and they affect physiology and protein evolution. Mapping translation errors in proteomes and understanding their causes is hindered by lack of a proteome-wide experimental methodology. We present the first methodology for systematic detection and quantification of errors in entire proteomes. Following proteome mass spectrometry, we identify, in E. coli and yeast, peptides whose mass indicates specific amino acid substitutions. Most substitutions result from codon-anticodon mispairing. Errors occur at sites that evolve rapidly and that minimally affect energetic stability, indicating selection for high translation fidelity. Ribosome density data show that errors occur at sites where ribosome velocity is higher, demonstrating a trade-off between speed and accuracy. Treating bacteria with an aminoglycoside antibiotic or deprivation of specific amino acids resulted in particular patterns of errors. These results reveal a mechanistic and evolutionary basis for translation fidelity., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The localization of mRNAs encoding secreted/membrane proteins (mSMPs) to the endoplasmic reticulum (ER) likely facilitates the co-translational translocation of secreted proteins. However, studies have shown that mSMP recruitment to the ER in eukaryotes can occur in a manner that is independent of the ribosome, translational control, and the signal recognition particle, although the mechanism remains largely unknown. Here, we identify a cis-acting RNA sequence motif that enhances mSMP localization to the ER and appears to increase mRNA stability, and both the synthesis and secretion of secretome proteins. Termed SECReTE, for secretion-enhancing cis regulatory targeting element, this motif is enriched in mRNAs encoding secretome proteins translated on the ER in eukaryotes and on the inner membrane of prokaryotes. SECReTE consists of ≥10 nucleotide triplet repeats enriched with pyrimidine (C/U) every third base (i.e. NNY, where N = any nucleotide, Y = pyrimidine) and can be present in the untranslated as well as the coding regions of the mRNA. Synonymous mutations that elevate the SECReTE count in a given mRNA (e.g. SUC2, HSP150, and CCW12) lead to an increase in protein secretion in yeast, while a reduction in count led to less secretion and physiological defects. Moreover, the addition of SECReTE to the 3'UTR of an mRNA for an exogenously expressed protein (e.g. GFP) led to its increased secretion from yeast cells. Thus, SECReTE constitutes a novel RNA motif that facilitates ER-localized mRNA translation and protein secretion., Competing Interests: The authors have declared that no competing interests exist.

In experimental evolution, scientists evolve organisms in the lab, typically by challenging them to new environmental conditions. How best to evolve a desired trait? Should the challenge be applied abruptly, gradually, periodically, sporadically? Should one apply chemical mutagenesis, and do strains with high innate mutation rate evolve faster? What are ideal population sizes of evolving populations? There are endless strategies, beyond those that can be exposed by individual labs. We therefore arranged a community challenge, Evolthon, in which students and scientists from different labs were asked to evolve Escherichia coli or Saccharomyces cerevisiae for an abiotic stress-low temperature. About 30 participants from around the world explored diverse environmental and genetic regimes of evolution. After a period of evolution in each lab, all strains of each species were competed with one another. In yeast, the most successful strategies were those that used mating, underscoring the importance of sex in evolution. In bacteria, the fittest strain used a strategy based on exploration of different mutation rates. Different strategies displayed variable levels of performance and stability across additional challenges and conditions. This study therefore uncovers principles of effective experimental evolutionary regimens and might prove useful also for biotechnological developments of new strains and for understanding natural strategies in evolutionary arms races between species. Evolthon constitutes a model for community-based scientific exploration that encourages creativity and cooperation., Competing Interests: The authors have declared that no competing interests exist.

The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained. We identified two distinct and synergistic transcriptional modules that dominate successful reprogramming, which are associated with cell identity and biosynthetic genes. The pluripotency module is governed by dynamic alterations in epigenetic modifications to promoters and binding by Oct4, Sox2, and Klf4, but not Myc. Early DNA demethylation at certain enhancers prospectively marks cells fated to reprogram. Myc activity drives expression of the essential biosynthetic module and is associated with optimized changes in tRNA codon usage. Our functional validations highlight interweaved epigenetic- and Myc-governed essential reconfigurations that rapidly commission and propel deterministic reprogramming toward naive pluripotency., (Copyright © 2018 Elsevier Inc. All rights reserved.)