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A progressive loss of protein homeostasis is characteristic of aging and a driver of neurodegeneration. To investigate this process quantitatively, we characterized proteome dynamics during brain aging in the short‐lived vertebrate Nothobranchius furzeri combining transcriptomics and proteomics. We detected a progressive reduction in the correlation between protein and mRNA, mainly due to post‐transcriptional mechanisms that account for over 40% of the age‐regulated proteins. These changes cause a progressive loss of stoichiometry in several protein complexes, including ribosomes, which show impaired assembly/disassembly and are enriched in protein aggregates in old brains. Mechanistically, we show that reduction of proteasome activity is an early event during brain aging and is sufficient to induce proteomic signatures of aging and loss of stoichiometry in vivo . Using longitudinal transcriptomic data, we show that the magnitude of early life decline in proteasome levels is a major risk factor for mortality. Our work defines causative events in the aging process that can be targeted to prevent loss of protein homeostasis and delay the onset of age‐related neurodegeneration. Synopsis Analyses of proteome dynamics delineate a timeline of molecular events underlying brain aging in the vertebrate Nothobranchius furzeri . Early‐in‐life decline of proteasome activity is associated with loss of stoichiometry of protein complexes and predicts lifespan. Progressive loss of stoichiometry affects multiple protein complexes. Ribosomes aggregate in old brains. Partial reduction of proteasome activity is sufficient to induce loss of stoichiometry. Reduced proteasome levels are a major risk factor for early death in killifish. Graphical Abstract Analyses of proteome dynamics delineate a timeline of molecular events underlying brain aging in the vertebrate Nothobranchius furzeri . Early‐in‐life decline of proteasome activity is associated with loss of stoichiometry of protein complexes and predicts lifespan.

Understanding how the homeostasis of cellular size and composition is accomplished by different organisms is an outstanding challenge in biology. For exponentially growing Escherichia coli cells, it is long known that the size of cells exhibits a strong positive relation with their growth rates in different nutrient conditions. Here, we characterized cell sizes in a set of orthogonal growth limitations. We report that cell size and mass exhibit positive or negative dependences with growth rate depending on the growth limitation applied. In particular, synthesizing large amounts of “useless” proteins led to an inversion of the canonical, positive relation, with slow growing cells enlarged 7‐ to 8‐fold compared to cells growing at similar rates under nutrient limitation. Strikingly, this increase in cell size was accompanied by a 3‐ to 4‐fold increase in cellular DNA content at slow growth, reaching up to an amount equivalent to ~8 chromosomes per cell. Despite drastic changes in cell mass and macromolecular composition, cellular dry mass density remained constant. Our findings reveal an important role of protein synthesis in cell division control. Synopsis Protein overexpression inverts the well‐known positive relation of cell size and DNA content with growth rate under nutrient limitation, resulting in huge, slowly growing cells with high DNA content, but with remarkably constant cellular dry mass density. Protein overexpression leads to an inverted growth rate dependence of cell size, with slow growing cells being even larger than the fastest growing wild‐type cells cultured in rich media. Decoupled from growth rate, changes in cell size are accompanied by comparable changes in cellular DNA content. Despite profound changes in cell size and macromolecular composition, cellular dry mass density remains remarkably constant, as protein overexpression does not result in molecular crowding. Graphical Abstract Protein overexpression inverts the well‐known positive relation of cell size and DNA content with growth rate under nutrient limitation, resulting in huge, slowly growing cells with high DNA content, but with remarkably constant cellular dry mass density.

Functional annotation of proteins is a fundamental problem in the post‐genomic era. The recent availability of protein interaction networks for many model species has spurred on the development of computational methods for interpreting such data in order to elucidate protein function. In this review, we describe the current computational approaches for the task, including direct methods, which propagate functional information through the network, and module‐assisted methods, which infer functional modules within the network and use those for the annotation task. Although a broad variety of interesting approaches has been developed, further progress in the field will depend on systematic evaluation of the methods and their dissemination in the biological community.

Proper functioning of biological cells requires that the process of protein expression be carried out with high efficiency and fidelity. Given an amino‐acid sequence of a protein, multiple degrees of freedom still remain that may allow evolution to tune efficiency and fidelity for each gene under various conditions and cell types. Particularly, the redundancy of the genetic code allows the choice between alternative codons for the same amino acid, which, although ‘synonymous,’ may exert dramatic effects on the process of translation. Here we review modern developments in genomics and systems biology that have revolutionized our understanding of the multiple means by which translation is regulated. We suggest new means to model the process of translation in a richer framework that will incorporate information about gene sequences, the tRNA pool of the organism and the thermodynamic stability of the mRNA transcripts. A practical demonstration of a better understanding of the process would be a more accurate prediction of the proteome, given the transcriptome at a diversity of biological conditions.

Profiling of biological relationships between different molecular layers dissects regulatory mechanisms that ultimately determine cellular function. To thoroughly assess the role of protein post‐translational turnover, we devised a strategy combining pulse stable isotope‐labeled amino acids in cells (pSILAC), data‐independent acquisition mass spectrometry (DIA‐MS), and a novel data analysis framework that resolves protein degradation rate on the level of mRNA alternative splicing isoforms and isoform groups. We demonstrated our approach by the genome‐wide correlation analysis between mRNA amounts and protein degradation across different strains of HeLa cells that harbor a high grade of gene dosage variation. The dataset revealed that specific biological processes, cellular organelles, spatial compartments of organelles, and individual protein isoforms of the same genes could have distinctive degradation rate. The protein degradation diversity thus dissects the corresponding buffering or concerting protein turnover control across cancer cell lines. The data further indicate that specific mRNA splicing events such as intron retention significantly impact the protein abundance levels. Our findings support the tight association between transcriptome variability and proteostasis and provide a methodological foundation for studying functional protein degradation. Synopsis This study analyzes the gene isoform‐specific relationships between mRNA variation and protein degradation and underscores the diversity of protein turnover control in steady‐state gene expression. An optimized experimental and bioinformatic workflow is developed to study protein turnover in high throughput by combining single‐shot Data independent acquisition mass spectrometry (DIA‐MS) and pulse‐chase SILAC labeling experiments. The genome‐wide, protein specific correlation between mRNA variation and protein degradation is a powerful measure of post‐translational control in determining protein variability. The correlation analysis reveals the diversity of protein turnover at various scales, ranging from specific biological processes and organelles to sub‐organelles and splicing isoforms. mRNA intron retention switching mostly impacts the corresponding protein abundance but not protein degradation. Graphical Abstract This study analyzes the gene isoform‐specific relationships between mRNA variation and protein degradation and underscores the diversity of protein turnover control in steady‐state gene expression.

Different tissues express genes with particular codon usage and anticodon tRNA repertoires. However, the codon–anticodon co‐adaptation in humans is not completely understood, nor is its effect on tissue‐specific protein levels. Here, we first validated the accuracy of small RNA‐seq for tRNA quantification across five human cell lines. We then analyzed the tRNA abundance of more than 8,000 tumor samples from TCGA, together with their paired mRNA‐seq and proteomics data, to determine the Supply‐to‐Demand Adaptation. We thereby elucidate that the dynamic adaptation of the tRNA pool is largely related to the proliferative state across tissues. The distribution of such tRNA pools over the whole cellular translatome affects the subsequent translational efficiency, which functionally determines a condition‐specific expression program both in healthy and tumor states. Furthermore, the aberrant translational efficiency of some codons in cancer, exemplified by ArgAGA, is associated with poor patient survival † . The regulation of these tRNA profiles is partly explained by the tRNA gene copy numbers and their promoter DNA methylation. Synopsis Quantification of tRNA expression over thousands of small RNA‐seq samples from The Cancer Genome Atlas unveils the existence of tissue‐specific translational efficiencies related to proliferation. tRNA abundance is tissue‐ and cancer‐type-specific. Translational efficiency is globally controlled and the cellular translatome needs to compete for a limiting tRNA pool. Proliferation is the major determinant of translational efficiency differences among tissues, and the codon ProCCA appears particularly favored in cancer. Differences at the tRNA pool affect protein translation and subsequently determine specific functional phenotypes. Graphical Abstract Quantification of tRNA expression over thousands of small RNA‐seq samples from The Cancer Genome Atlas unveils the existence of tissue‐specific translational efficiencies related to proliferation.

Transcription initiated at alternative sites can produce mRNA isoforms with different 5ʹUTRs, which are potentially subjected to differential translational regulation. However, the prevalence of such isoform‐specific translational control across mammalian genomes is currently unknown. By combining polysome profiling with high‐throughput mRNA 5ʹ end sequencing, we directly measured the translational status of mRNA isoforms with distinct start sites. Among 9,951 genes expressed in mouse fibroblasts, we identified 4,153 showed significant initiation at multiple sites, of which 745 genes exhibited significant isoform‐divergent translation. Systematic analyses of the isoform‐specific translation revealed that isoforms with longer 5ʹUTRs tended to translate less efficiently. Further investigation of cis‐ elements within 5ʹUTRs not only provided novel insights into the regulation by known sequence features, but also led to the discovery of novel regulatory sequence motifs. Quantitative models integrating all these features explained over half of the variance in the observed isoform‐divergent translation. Overall, our study demonstrated the extensive translational regulation by usage of alternative transcription start sites and offered comprehensive understanding of translational regulation by diverse sequence features embedded in 5ʹUTRs. Synopsis Polysome profiling combined with 5ʹ‐end sequencing in mouse fibroblasts shows pervasive isoform‐specific translational regulation via alternative transcription start sites (TSSs) and reveals 5′UTR sequence features linked to translational regulation. Isoform‐specific translational regulation is achieved through alternative TSS usage. Isoforms with longer 5ʹUTRs tend to have lower translational efficiency (TE). Systematic analyses of isoform‐specific translation offer new insights into the regulation by known sequence features and identifies novel regulatory sequence motifs. Quantitative models integrating all identified sequence features explain over half of the variance in the observed TE divergence between isoforms. Graphical Abstract Polysome profiling combined with 5ʹ‐end sequencing in mouse fibroblasts shows pervasive isoform‐specific translational regulation via alternative transcription start sites (TSSs) and reveals 5′UTR sequence features linked to translational regulation.

Translation efficiency has been mainly studied by ribosome profiling, which only provides an incomplete picture of translation kinetics. Here, we integrated the absolute quantifications of tRNAs, mRNAs, RNA half‐lives, proteins, and protein half‐lives with ribosome densities and derived the initiation and elongation rates for 475 genes (67% of all genes), 73 with high precision, in the bacterium Mycoplasma pneumoniae ( Mpn ). We found that, although the initiation rate varied over 160‐fold among genes, most of the known factors had little impact on translation efficiency. Local codon elongation rates could not be fully explained by the adaptation to tRNA abundances, which varied over 100‐fold among tRNA isoacceptors. We provide a comprehensive quantitative view of translation efficiency, which suggests the existence of unidentified mechanisms of translational regulation in Mpn . Synopsis Integration of the absolute quantifications of mRNAs, RNA half‐lives, proteins, protein half‐lives, tRNAs, with ribosome densities offers a comprehensive quantitative study of translation efficiency in the genome‐reduced bacterium Mycoplasma pneumoniae ( Mpn ). Integration of the absolute quantifications of mRNAs, proteins, and protein half‐lives with ribosome densities allowed to derive the initiation and elongation rates for 475 genes (67% of all genes) in the genome‐reduced bacterium Mpn . Translation initiation rate varies over 160‐fold among genes, and most of the known factors have little impact on translation efficiency. Measured tRNA abundances vary over 100‐fold among tRNA isoacceptors, and this variation could not explain local codon elongation rates. A comprehensive quantitative study of translation efficiency suggests the existence of unidentified mechanisms of translational regulation in Mpn . Graphical Abstract Integration of the absolute quantifications of mRNAs, RNA half‐lives, proteins, protein half‐lives, tRNAs, with ribosome densities offers a comprehensive quantitative study of translation efficiency in the genome‐reduced bacterium Mycoplasma pneumoniae ( Mpn ).

The C‐terminal sequence of a protein is involved in processes such as efficiency of translation termination and protein degradation. However, the general relationship between features of this C‐terminal sequence and levels of protein expression remains unknown. Here, we identified C‐terminal amino acid biases that are ubiquitous across the bacterial taxonomy (1,582 genomes). We showed that the frequency is higher for positively charged amino acids (lysine, arginine), while hydrophobic amino acids and threonine are lower. We then studied the impact of C‐terminal composition on protein levels in a library of Mycoplasma pneumoniae mutants, covering all possible combinations of the two last codons. We found that charged and polar residues, in particular lysine, led to higher expression, while hydrophobic and aromatic residues led to lower expression, with a difference in protein levels up to fourfold. We further showed that modulation of protein degradation rate could be one of the main mechanisms driving these differences. Our results demonstrate that the identity of the last amino acids has a strong influence on protein expression levels. Synopsis Large‐scale genomics analyses combined with high‐throughput experimental assays reveal that the C‐terminal amino acid composition has a strong influence on protein expression levels in bacteria. C‐terminal amino acid biases are ubiquitous across bacterial taxonomy: positively charged residues (lysine, arginine) are enriched at the last position, while hydrophobic amino acids and threonine are depleted. High‐throughput expression assays using a reporter gene library showed that protein expression varies up to 4‐fold, with C‐terminal positively and negatively charged residues increasing expression, and hydrophobic residues decreasing expression. Modulation of protein degradation rate due to the identity of the C‐terminal residue could explain ˜ 85% of the variation in protein expression. These results are relevant for the optimization of heterologous protein sequences, where the choice of C‐terminal residues could lead to increased expression levels. Graphical Abstract Large‐scale genomics analyses combined with high‐throughput experimental assays reveal that the C‐terminal amino acid composition has a strong influence on protein expression levels in bacteria.

Purely in vitro ribosome synthesis could provide a critical step towards unraveling the systems biology of ribosome biogenesis, constructing minimal cells from defined components, and engineering ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli ribosome reconstitution are non‐physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one‐step co‐activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional ribosomes, and synthesis of active protein by these ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of ribosome assembly and open new avenues for making ribosomes with altered properties. This report describes an integrated method for in vitro construction of Escherichia coli ribosomes under near‐physiological conditions. This method enables coupling of ribosome synthesis and assembly in a single, integrated system. Synopsis This report describes an integrated method for in vitro construction of Escherichia coli ribosomes under near‐physiological conditions. This method enables coupling of ribosome synthesis and assembly in a single, integrated system. An integrated synthesis, assembly, and translation technology (termed iSAT) was developed to construct ribosomes in vitro . iSAT mimics co‐transcription of rRNA and ribosome assembly as it occurs in vivo . iSAT makes possible the in vitro construction of modified ribosomes. iSAT is expected to aid studies of ribosome assembly and open new avenues for making ribosomes with altered capabilities.